In vivo characterization

of Biomaterials

TEB 2020-2021
Soledad Perez Amodio
§ In vivo testing of Biomaterials

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§ Why in vivo testing?
Limitations of in vitro testing:

§ They cannot recreate the complex in vivo environment

§ Immune reaction
§ Functional properties of the scaffold can not be studied
§ Role of angiogenesis in newly formed tissue

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§ Animal Models

Living organism that in one or more anatomical or physiological aspects

can be compared with the healthy or diseased man because of the reaction
upon external stimuli.

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In vivo tests
§ Sensitization (ISO 10993-10:2010): guinea pig maximization test
(GPMT), guinea pig closed patch or Buehler and local lymph node assay
(LLNA).

§ Irritation (ISO 10993-10:2010): skin, ocular, mucous membrane and

intracutaneous assays.

§ Systemic toxicity (ISO 10993-11:2006): acute, subacute, sub- chronic,

and chronic

§ Implantation (ISO 10993-6:2007): muscle, subcutaneous tissue, bone.

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§ Choosing the animal model

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Animal models

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Rodents (Rats and Mice)
§ Widely used in all fields of tissue engineering research.
§ Moderate costs and ease of care.
§ Study of biological features of cells and engineered tissues,
preliminary proof-of-concept experiments.
§ Commercially available rodent strains are highly standardized.
§ Due to the small size of the animals surgical procedures are
difficult to perform: results cannot easily be transferred to
humans.

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Pigs
§ Similar anatomy, physiology and immunology to that of
humans
§ Uncooperative with full general anaesthetic
§ Capable of rapid growth: limited time TE construct
assessment
§ Surgical and anaesthesiologic skills required to perform
operations
§ Strain to reduce final body size and rate of growth: Minipigs

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Dogs

§ Convenient size (10–30 kg), ease of care and modest growth

rates.

§ Ethical considerations raised particularly in Western countries.

§ Dogs bred specifically for experimental research: beagles,

foxhounds, Labradors.

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Rabbits

§ Ready available, relatively cheap and easy to maintain.

§ Surgical procedures difficult as compared with larger animals.

§ Comparable to other rodents, the transfer of research results to humans

is not be easily accomplished.

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Primates
§ Apes and monkeys: the animal model most closely resembling human
anatomy and physiology.
§ Slow and predictable rate of growth: suitable for experiments
determining the long-term behaviour of tissue engineered grafts.
§ Ethical and legal restrictions.
§ Excessive costs in care and feeding.

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§ Costs of animal models

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§ Advantages and dissadvantages of in vivo animal models

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Examples of in vivo testing biomaterials

§ Inflammation and host reaction

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§ Bone regeneration § Cartilage repair

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§ Non-invasive in vivo characterization of biomaterials

In vivo optical bioluminescence imaging (BLI)

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Zebrafish embryo
§ Optical transparency allows real time visualization
§ Suitable for studying innate response: innate immune system similar to mammals
§ High fertility: large number of embryos
§ Easy and cheap maintenance: 100-fold reduced cost than for mice

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Transgenic Zebrafish

Macrophages (green)
Neutrophils (red).

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Chicken embryo
§ Chick Chorioallantoic membrane (CAM) assay

Vascular membrane found in eggs of birds and reptiles.

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Chicken embryo development

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Advantages of CAM model

§ Highly vascularized nature

§ High reproducibility

§ Simplicity

§ Cost-and time- efficient

§ Naturally immunodeficient host

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IN-ovo and ex-ovo CAM assay
In-ovo Ex-ovo

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IN-ovo and ex-ovo CAM assay

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§ Angiogenic capacity of biomaterials

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§ Ethical issues

Unhatched birds are not considered living animals by

national legislations worldwide

Experiments performed on avian embryos seem to be

ethically more tolerable than those on rodents

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ANIMAL WELFARE

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Ethical concerns relevant to the use of animals in Tissue Engineering
§ Is the animal model relevant to human physiology?

§ Is the experiment well-designed and outcome sufficiently important so

that the data obtained will justify the suffering and sacrifice of the life
of a living creature?

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§ Legislation for the protection of animals used for research

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§ Before experiments on animals are conducted, the research protocol must be
reviewed by animal ethics committees. The guiding principle of these
committees is usually the 3 Rs.
§ All researchers handling the animals used for experimentation should also be
trained in handling the particular species in the study

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Animal welfare and the three Rs

The guiding principles supporting the humane

use of animals:

§ Replacement
§ Reduction
§ Refinement

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§ Replacement

Animal experiments must be replaced wherever possible

by other methods.

§ Statistical data already obtained from animal research

§ Studies of isolated cells and tissues
§ Computer models that simulate an animal’s response to specific
experiments
§ Studies of patients and populations

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Examples

§ Test using bacteria instead of rodents to see whether a chemical is

likely to damage DNA or has potential to cause cancer.

§ Scientists can check injected medicines for most pyrogens (fever-

causing bacterial contaminants) by using blood cells– replacing
tests on rabbits.

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§ Refinement

§ Refine the way experiments are carried out.

§ Better housing.

§ Improvements to procedures which minimise pain and suffering.

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§ Examples

§ Food rewards can be used to train monkeys to sit on weighing scales,

or hold out a limb to give a blood sample – reducing any stress.

§ Blood pressure, heart rate and activity levels can be measured

by radio-operated implants, so that animals do not have to be
repeatedly caught or restrained.

§ Rodents can be housed in a special red plastic ‘house’ so they

feel they’re nesting in a dark place (they can’t see through red), yet
it allows them to be seen and studied.

§ Animals are routinely kept in social groups and given stimulating

environments.
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§ Reduction

§ Reduce the number of animals used to a minimum.

§ Obtain information from fewer animals.

§ Obtain more information from the same number of animals.

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§ Examples

§ Statistical analysis ensure that researchers use the

optimum number of animals.

§ Using inbred animals means researchers can get reliable

results from fewer of them.

§ New scanning techniques: tumours can be

tracked non-invasively (more data collected from the same animal).

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Alternative to animal testing
§ TE for modelling human physiology
Creation of in vitro human models to identify the factors that drive cellular processes

Skin wound healing

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§ Organ-on-a-chip

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 § Computer (in silico) Modeling

Simulated prevascularized scaffolds

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§ Cellular in vitro models

Cancer models 3D hepatocyte cultures

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§ Organoids

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§ Bioprinted tissues

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