Biocompatibilit

y

Testing

Swapnil Singh, NIPER, Mohali

IntroductionAcceptance of an artificial implant by the surrounding tissues and by the body as a whole

The ability of a material to perform its desired function with respect to a medical therapy, without eliciting any undesirable local or systemic effects in the recipient or beneficiary of that therapy, but generating the most appropriate beneficial cellular or tissue response in that specific situation, and optimizing the clinically relevant performance of that therapy (Williams, 2008)

A material is considered “biocompatible” if it allows the body to function without complications like allergic reactions or adverse side effects. Biocompatibility is the “suitability” of a material for exposure to the body or bodily fluids

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When lacking biocompatiblity…

long-lasting chronic inflammation

cytotoxic chemicals

disruption of cells at interface

micron-sized materials

irritation

corrosion of metals

restenosis/thrombosis

Plan of biocompatibility tests

in vitro tests Animal experiments Clinical tests

•Evaluation under in vitro (literally “in glass”) conditions can provide rapid and inexpensive data on biological interaction

•Will the in vitro test measure parameters relevant to what will occur in the much more complex in vivo environment?

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

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Contd..

•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

•The common approach is to start with simple in vitro tests

•If these experiments and investigations of a material’s efficiency deliver promising findings, then more comprehensive studies on experimental animals (in vivo evaluation) will be performed

•Clinical trials are the final step of this evaluation process

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in vitro tests•Less expensive way

•Simulate biological reactions to materials when they are placed on or into tissues of the body

ADVANTAGE• Experimentally controllable, repeatable, • Fast, relatively inexpensive, relatively simple.• Avoid the ethical and legal issues• Transgenic cells carrying human genes can be used• Small amount of test material is required

DISADVANTAGE• Questionable clinical relevance• Chronic effects cannot be tested• Pharmacokinetics cannot be evaluated

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in vivo tests •The goal is to determine the biocompatibility or safety in a biological environment

•Carried out to determine that the device performs as intended and presents no significant harm to the patient or user

ADVANTAGES• Higher level of significance • Simulate real body conditions

DISADVANTAGES• Expensive• Time consuming • Ethical issues 5

Factors considered during in vivo test

•Chemical composition of the materials

•Nature, degree, frequency, and

•Duration of exposure of the device and its constituents to the intended tissues

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Selection of in vivo tests

To facilitate the selection of appropriate tests biomaterials can be categorized by

Nature of body contact of the medical device and by

Duration of contact of the medical device

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TABLE 1 Medical Device Categorization by Tissue Contact and Contact Duration

Surface devices SkinMucosal membranesBreached or compromised surfaces

External communicatingdevices

Blood path, indirectTissue/bone/dentin communicatingCirculating blood

Implant devices Tissue/boneBlood

Contact duration Limited, ≤24 hoursProlonged, >24 hours and <30 daysPermanent, >30 days

Two perspectives in in vivo testing

1. Utilization of in vivo tests to determine the general biocompatibility of newly developed biomaterials for which some knowledge of the tissue compatibility is necessary for further research and development

2. It focuses on the biocompatibility of the final product, that is, the medical device in the condition in which it is to be implanted

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Various in vivo Tests as per ISO 10993 standard and the FDA guidance document are:

in vivo tests

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ISO 10,993-1. Evaluation and testingISO 10,993-2. Animal welfare requirementsISO 10,993-3. Tests for genotoxicity, carcinogenicity,and reproductive toxicityISO 10,993-4. Selection of tests for interactionswith bloodISO 10,993-5. Tests for cytotoxicity: In vitro methodsISO 10,993-6. Tests for local effects after implantationISO 10,993-7. Ethylene oxide sterilization residualsISO 10,993-9. Framework for the identificationand quantification of potentialdegradation productsISO 10,993-10. Tests for irritation and sensitizationISO 10,993-11. Tests for systemic toxicityISO 10,993-12. Sample preparation and referencematerialsISO 10,993-13. Identification and quantification ofdegradation products from polymersISO 10,993-14. Identification and quantification ofdegradation products from ceramicsISO 10,993-15. Identification and quantification ofdegradation products from metalsand alloysISO 10,993-16. Toxicokinetic study design fordegradation products and leachables

ISO 10,993, Biological Evaluation of Medical Devices, International Standards Organization

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Sensitization test•Allergic response caused by the activation of complex cellular and humoral immunological mechanisms can occur after either single or multiple exposures

•Animal used: Guinea pig

•Method: Buehler closed-patch test and the Magnusson-Kligman guinea pig maximization (ISO 10993-10)

•If the test material is amenable to intradermal injection, the maximization test is recommended. The closed-patch test is the assay of choice for non-extractable, or when the extract or material may be topically applied

•Induction phase followed by challange phase

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Systemic toxicity •It 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, biomaterials, and/or their extracts

•Conducted by administering the extracts (polar and nonpolar in most cases) as a single dose to test animals, and the health status of the animals is verified periodically—typically 24, 48 and 72 hours after dosing

•Solvents, should be chosen to yield a maximum extraction of leachable materials for use in the testing

• Animals of choice for the conduct of these tests are mice, rats, or rabbits

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• It may be acute, sub-acute or chronic

• 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

• 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

Contd...

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ISO defines the clinical observation descriptors as respiratory, motor activities, convulsion, reflexes, ocular signs, cardiovascular signs, salivation, piloerection, analgesia, muscle tone, gastrointestinal and skin

Contd...RESPONSE DESCRIPTION

Normal, no symptoms No adverse physical symptoms after injection

Slight Slight loss of motor function, slight difficulty breathing, and symptoms of irritation in the abdominal cavity

Moderate Difficulty breathing, loss of motor function, dropping of eyelids, and diarrhea clearly observed

Marked Cyanosis, and trembling, or a sever case of irritation in the abdominal cavity, diarrhea, drooping of the eyelids, and difficulty of breathing are observed

Dead, expired Mouse dies after injection

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

•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, and

Chromosomal aberrations

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in vivo genotoxicity

tests

chromosomal analysis

mouse spot test

micronucleus test

mouse heritable

translocation assay

mammalian germ cell

cytogenetic assay

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The most common test is the rodent micronucleus test:

• The in vivo test normally uses mouse bone marrow or

mouse peripheral blood

• Micronuclei, also known as Howell–Jolly bodies, are

generally smooth, round remnants of nuclear

chromatin seen in erythrocytes

• An increase in the frequency of micronucleated

erythrocytes in treated animals is an indication of

induced chromosome damage

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Micronucleated erythrocytes

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Implantation test

• Implantation tests assess the local pathological effects on the structure and function of living tissue induced by a sample of a material or final product

• For short-term implantation evaluation out to 12 weeks

• Animals utilized in these studies are mice, rats, guinea pigs, or rabbits

• For longer-term testing in subcutaneous tissue, muscle, or bone • Animals such as rabbits, dogs, sheep, goats, pigs, and

other animals with relatively long life expectancy are suitable

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Contd...

• Short-term effects are assessed by evaluating tissue

responses to the implant at 1 and 4 weeks following the

procedure

• At least four rabbits per time period are recommended,

and each rabbit is implanted with at least four test and

two negative control materials

• Evaluated responses include inflammatory reactions and

the area thus affected

• If at least two of the four test sites exhibit a significant

response compared with the control sites, an adverse or

positive effect can be assumed

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Eye irritation test (Draize test)• Local tissue inflammation response to chemicals, without

a systemic immunological component.

• Test animal: Rabbit

• Volume of Extract: 0.1 mL

• Extract is instilled in one eye of each animal, the other

eye receives the control vehicle

• The test and control eyes are assessed for biological

responses at 1, 24, 48, and 72 hours after instillation

• The observation period need not exceed 21 days

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SCORE OBSERVED EFFECT

0 Non irritant

0-0.5 Minimal irritant

0.5-2 Mild irritant

2-5 Moderate irritant

5-8 Severe irritant

Contd...

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Skin irritation test

• Measured by Trans-epithelial Water Loss (TEWL) test

• TEWL measurements are of great importance in

evaluating barrier functionality

• The more perfect the skin protective coat, the higher

the water content and the lower the TEWL

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Figure 1. Schematic illustration of the barrier function of the stratum corneum. a) healthy skin , b) disturbed skin

• Pyrogens are substances in devices that cause a febrile

reaction

• Bacterial endotoxin contamination is most commonly

associated with such an adverse effect; however,

leachates of materials can cause similar febrile responses

(material-mediated pyrogenicity)

• ISO 10993-11 recommends testing the pyrogenicity

potential of extractable substances derived from material

leaching

Pyrogen testing

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Number of animals: Three rabbits required; comparison of febrile response in test animals to baseline temperature for evaluation of pyrogenicity potential

Test duration: Test measurement intervals: every 30 minutes for 3 hours

Evaluation: Cutoff for positive febrile response: 0.5°C

If any single animal of the three has a temperature increase above the acceptable range, the test can be continued with 5 additional animals

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Carcinogenicity

• Carcinogens induce tumors (benign or malignant),

increase their incidence or malignancy, or shorten the

time of tumor occurrence when they are inhaled,

injected, dermally applied, or ingested

• This test determine the tumorigenic potential of

medical devices, materials, and/or their extracts from

either single or multiple exposures

Maurici, et al., 2005, p. 177

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• Carcinogenicity tests should be conducted only if data

from other sources suggest a tendency for tumor

induction

• The conventional test for carcinogenicity is the long-

term rodent carcinogenicity bioassay requiring 2 years

• Carcinogenicity and chronic toxicity may be studied in a

single experimental study

• To facilitate and reduce the time period for

carcinogenicity testing of biomaterial, the FDA is

exploring the use of transgenic mice carrying the human

prototype c-Ha-ras gene as a bioassay mode for rapid

carcinogenicity testing.

Contd...

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• The gene, is capable of transforming normal cells into a neoplastic cell following its mutation, confers an unusually high susceptibility to tumor formation in rasH2 transgenic mice

• Various advantages of c-Ha-ras animals are:

Contd...

Mutagen detection within 6 months

Able to detect various non mutagenic carcinogens

More rapid onset and higher incidence of tumors

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Hemocompatibility

BIOMATERIAL

BLOOD

HUMORAL EFFECTSCELLULAR EFFECTSex: thrombosis, embolism, lysis, and inflammation

ex: activation of the coagulation, kinin, complement, and fibrinolytic systems

Device contact with circulating blood

DIRECT INDIRECTIMPLANTSDrainage catheters, Butterfly needles

Stents, Cardiac valve Blood bag

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Platelets adhesion and aggregation

Thrombosis

• Thrombosis is the formation of a blood clot inside a blood vessel, obstructing the flow of blood through the circulatory system

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Test article (e.g., tubing or catheter) is implanted in the

jugular veins of two (2) dogs.

The test article is implanted in the jugular vein

The test article are removed and examined for the

presence of thrombi, and the vein is examined for

patency (occlusion)

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Possible scenarios for blood–materials interactions

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THANK YOU

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