The document discusses biocompatibility, emphasizing the need for materials to function safely within the body without adverse reactions. It outlines various testing methodologies including in vitro and in vivo tests for evaluating biocompatibility, detailing their advantages, disadvantages, and specific tests per ISO standards. Key testing parameters also include acute and chronic toxicity, genotoxicity, and hemocompatibility, with protocols for testing and evaluation to ensure safety in medical devices.

1. Swapnil Singh, NIPER,
Mohali

2. Introduction
Acceptance 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
1

3.  When lacking biocompatiblity…
 long-lasting chronic inflammation
 cytotoxic chemicals
 disruption of cells at interface
 micron-sized materials
 irritation
 corrosion of metals
 restenosis/thrombosis

4. 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
2

5. 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
3

6. 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
4

7. 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

8. 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
6

9. 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
7

10. TABLE 1 Medical Device Categorization by Tissue Contact
and Contact Duration
Surface devices Skin
Mucosal membranes
Breached or compromised
surfaces
External communicating
devices
Blood path, indirect
Tissue/bone/dentin
communicating
Circulating blood
Implant devices Tissue/bone
Blood
Contact duration Limited, ≤24 hours
Prolonged, >24 hours and
<30 days
Permanent, >30 days

11. 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
10

12. Various in vivo Tests as per ISO 10993 standard and the
FDA guidance document are:
in vivo
tests
11

13. 13
ISO 10,993-1. Evaluation and testing
ISO 10,993-2. Animal welfare requirements
ISO 10,993-3. Tests for genotoxicity, carcinogenicity,
and reproductive toxicity
ISO 10,993-4. Selection of tests for interactions
with blood
ISO 10,993-5. Tests for cytotoxicity: In vitro methods
ISO 10,993-6. Tests for local effects after implantation
ISO 10,993-7. Ethylene oxide sterilization residuals
ISO 10,993-9. Framework for the identification
and quantification of potential
degradation productsISO 10,993-10. Tests for irritation and sensitization
ISO 10,993-11. Tests for systemic toxicity
ISO 10,993-12. Sample preparation and reference
materials
ISO 10,993-13. Identification and quantification of
degradation products from polymers
ISO 10,993-14. Identification and quantification of
degradation products from ceramics
ISO 10,993-15. Identification and quantification of
degradation products from metals
and alloys
ISO 10,993-16. Toxicokinetic study design for
degradation products and leachables
ISO 10,993, Biological Evaluation of Medical Devices,
International Standards Organization

14. 14

15. 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
13

16. 16

17. 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
14

18. • 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
18
Contd...

19.  ISO defines the clinical observation descriptors as
respiratory, motor activities, convulsion, reflexes,
ocular signs, cardiovascular signs, salivation,
piloerection, analgesia, muscle tone, gastrointestinal
and skin
19
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

20. 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

21. 21
in vivo
genotoxicity
tests
chromosomal
analysis
mouse spot
test
micronucleus
test
mouse
heritable
translocation
assay
mammalian
germ cell
cytogenetic
assay

22. 22
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

23. 23
Micronucleated erythrocytes

24. 24
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

25. 25
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

26. 26
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

27. 27

28. 28

29. 29
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...

30. 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
30

31. 31
Figure 1. Schematic illustration of the barrier function of
the stratum corneum. a) healthy skin , b) disturbed skin

32. •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

33.  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 33

34. 34
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

35. 35
• 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...

36. 36
• 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

37. Hemocompatibility
37
BIOMATERIAL
BLOOD
HUMORAL EFFECTSCELLULAR EFFECTS
ex: thrombosis,
embolism, lysis, and
inflammation
ex: activation of the
coagulation, kinin,
complement, and
fibrinolytic systems
Device contact with circulating blood
DIRECT INDIRECTIMPLANTS
Drainage catheters,
Butterfly needles
Stents, Cardiac valve Blood bag

38. 38

39. 39
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

40. 40
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)

41. 41
Possible scenarios for blood–materials interactions

42. 42
THANK YOU

43. 43

44. 44