Biological implant evaluation
Exam: mostly open questions.
Assignment: individual.
Lecture 1
15-3-2021
Definitions
Testing cells + toxins. It tests the cytotoxicity of an implant to cells surrounding the implant.
Biomaterials that make up these implants are very diverse. Surface and bulk characteristics can be
designed independently. Characteristics often can be tuned to a specific application. We need to look
for each application specific what is needed.
Biomaterial: a material intended to interface with the biological system to evaluate, treat, augment
or replace tissue, organ or function of the organism. However, a biomaterial can be part of our food
chain for example and it is by definition not always an implant. Definition by ISO of a biomaterial:
material specially prepared and/or presented to exhibit biocompatibility. The environment and the
biomaterial need to work together in a positive fashion. Definition by FDA: defined as any material
used in the body to achieve a therapeutic or diagnostic purpose. It can be used inside the human
body as/in: prostheses, matrix material for tissue engineered devices, a capsule for cellular implant.
It can be used outside in extra corporeal devices. Nanoparticles can also be considered biomaterial
and can be used to make things visible with imaging (like MRI etc.).
Definition of biocompatibility: capacity of a material to fulfil its function with an appropriate
response for a specific application from the receiving host; quality of being accepted in a specific
living environment without adverse or unwanted side effects → the 2nd definition is not talking about
function, but more about safety. Most recent definition: biocompatibility refers to the ability of a
biomaterial to perform its desired function with respect to a medical therapy, without eliciting any
undesirable local or systemic effect 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.
Biological evaluation mainly concerns biological safety.
Bifunctionality: the quality of a biomaterial that satisfies those functional specification for which it
was designed. Think of functional specification of aspects such as incorporation of a hip implant into
the bone tissue for example. How does the bone respond to this hip implant?
Cytotoxicity
ISO standard 10993-5 (cytotoxicity): extract testing, direct contact testing and indirect contact
testing.
(1) Principle of extract testing
The fluid with a red color (cell culture medium) and put in a
bunch of biomaterial → let it sit for an amount of time at body
temperature (37 degrees) → use the culture medium to
cultivate cells → cells grow on petri dish which may consist the
biomaterial → cytotoxic compounds (leach into the cells), then
the cells will behave differently. We can follow the behavior of
the cells and see whether that is changed by the biomaterial.
- Extract testing = a way to determine if compounds in
materials can leach out of the material and can reach
concentrations that are toxic to tissue cells. The
material is incubating in a red liquid. This color is
, chosen, because the liquid usually is a cell culture medium that contains all the necessary
ingredients to allow cell growth / proliferation.
➔ The material is being incubated in the cell culture medium for 24h, 48h or 72h at usually 37
degrees Celsius.
➔ After that, the medium is given to a cell layer that is adhering to / growing on the bottom of
a cell culture plate.
➔ Those cells are then exposed to this medium for again 24h, 28h or 72h.
➔ The response of the cells is monitored. If there are toxic compounds, the cells will respond to
this decreased metabolic activity and even cell death (necrosis).
o Sometimes higher temperatures and more/less time is used than the standard 37
degrees Celsius. There is a limit of temperature for the cell culture medium, because
the proteins can otherwise behave differently or die. We would already affect the
medium when the conditions more than 50 degrees approximately. That is why we
mostly work with 37 degrees. Extraction conditions can be:
≥ 24 hours → 37 degrees.
72 hours → 50 degrees.
24 hours → 70 degrees.
1 hour → 121 degrees.
Higher temperatures are usually associated with higher release of compounds from
material. more than 50 degrees will destroy components of the medium, which by
itself then creates sub-optimal conditions later on. So, with higher temperatures
other liquids are often used, like phosphate buffers, physiological salt solution or
even water.
o Materials need to be sterile. If there is bacteria, they will contaminate the cell culture
medium and thereby making it impossible to use for the subsequent exposure of
cells to this extract. Also after sterilization (if materials have been produced under
dirty conditions) bacterial products may be abundantly present. These are known to
activate tissue cells.
o Material preparation: how much material do you mix with the medium of the cells?
There are certain ratios in the standards. That depends on the material (a film with a
surface area, bulky material etc.). We need to meet a certain ratio in order to have a
valid test.
o Control
▪ Negative control: no cytotoxic reaction, spontaneous response of cells.
Choice of ISO: high density polyethylene. It is totally non-toxic. Also here it
fully depends on how the material is made, in this case chains of monomers
with an initiator. Mostly the monomers itself are toxic, so it depends on how
the polymer is being made to make sure that these are ‘washed out’ so there
are no leachable compounds left in the material used.
▪ Positive control: yields reproducible cytotoxic reaction. Used:
polyvinylchloride with bound stannous ions or latex. It is very toxic to our
cells.
Cytotoxicity – general considerations
It is preferable to evaluate medical devices in their final product form. The biological testing must
incorporate everything involved in making the device. Equally important to device biocompatibility
are the processes and materials used during manufacturing. For most devices, the use of fluid extract
of the test materials prepared in a fashion to mimic to exaggerate the expected clinical conditions is
the most appropriate technique for determining the potential effects of chemical leachables.
It is advisable to have a visual on the cells during experiments, to have a direct control on how they
are responding (so like with a microscope).
,In a cell culture we are looking at cells that are growing on a transparent material (with a surface
treatment). When they cover the whole surface, it is called confluent. A phase contrast microscope
will lead to contrast to see the cells.
MTT assay
= metabolic assay; quantitative test. MTT is a chemical that is taken up in the cell. In the cells,
mitochondria are present. With oxygen they make ATP as an energy source. The MTT is a compound
that is converted by the same enzymes that create ATP by using oxygen. MTT is taken up in the
metabolic route of the cell, that is why it is called a metabolic assay. What we measure is the
converted product, which has a certain color. MTT assay is a typical timeline of studying the cell
response to an extract solution (again, the basis for this solution is cell culture medium).
- Cells adhere to surface or TCPS (Tissue culture polystyrene) for 24 hours. TCPS is the classical
gold standard for culturing cells, i.e. the standard material in a cell culture lab. The cells
proliferate and grow in the culture medium to form a kind of monolayer.
- The normal medium is taken off the cells and the extract is added.
- These cells are exposed to extracts for 24, 48 or 72 h.
- After this incubation, the cell response is being evaluated with form example the compound
MTT is converted into a blue compound by the cells when they are metabolically active.
- The cells get incubated with MTT for 3 hours. If cells are damaged by the toxicity of these
compounds → will not convert a lot of MTT. The cells are less lively. This can be seen after
three hours by determining the amount of blue. It can tell whether the population is still
doing well.
Summarized: cells cultured on medium → exposed to extracts you want to test → if MTT is
converted, the cells are vital and thus not damaged by toxins in the extracts. This can be seen
quantitively. More blue = more MTT converted = more cells metabolically active = more cells
undamaged by extract.
The blue solid is dissolved can be measured using spectroscopy. A high color development means
that cells are doing well. The use of extracts can give valuable information when we transform into a
so-called serial dilution. We start with undiluted extracts, then dilute it 2x in normal medium, then
4x, 8x and finally 16x. this means that the 16x diluted extract, when toxic, will have less toxic
influence than the undiluted extract, so we can get an estimate about where the toxicity limits are
positioned.
Analysis of cellular response by all current cell biological methods can be done with a confocal
microscopes. Proteins can be seen on the surface or that are excreted to the outside world. The
behavior of the cells can be seen. It measures fluorescence and sometimes fluorescent markers are
used. GFP can be used as a protein. In this a gene is modificated that expresses the protein that
, needs to be marked. Another (easier) way is to use antibodies. So methods for coupling fluorescent
labels to specific proteins:
- Gene modification, where you add the sequence for a fluorescent protein. The gene is
making a protein when it goes through translation and if you want to modify it, you have to
add the sequence behind the same promotor for this fluorescent protein, you have a label
protein. The main one is GFP.
- Fluorescent ligands, if you can make a fluorescent ligand that responds to this specific
receptor, it would work as a label for the protein. If you can target the receptor, you can see
whether the protein is there.
- Antibodies, they are very specific for a certain protein. You can tag those antibodies
(in)directly with a fluorescent molecule. In that way, you can look at almost all proteins in a
cell, since there are for all different antibodies.
Boundary of toxicity: control. Set the limit at 70% of that value relative to the control. If it is below
that 70 percent of the control, it is not toxic. 70%-100%, it is toxic.
Testing relevance
Cytotoxicity is important for all materials. When there are new biomaterial used, the whole ISO
process has to be tested. Sometimes not all steps have to be taken (some biomaterials are already
used for a very long time, so there is a lot of knowledge about it, so for the implant not all steps have
to be taken regarding cytotoxicity for the specific application). Cytotoxicity is just a part of the
biocompatibility testing.
(2) (in)direct contact testing
Looking at how cells interacts with material. The cells are tested directly in contact with the
biomaterial. Indirect → the cells that are not in direct contact or are close to the material, but still
tested to see if there are leachables that go further into the tissue. Direct → cells ‘touch’ the
biomaterial directly.
The material to be investigated, is fabricated in a disc shape. It is sterilized and then put in a cell
culture well. It can have the exact size of the well, but often is a bit
smaller (see image). The cells can be applied and suspended in cell
culture medium. It is put on top of the disc. Due the gravity, the cells
sink to the bottom and start to interact with the material. This is
direct contact. The cells that sink next to the disc on the well surface,
interact with well surface (which is TCPS), so cells like to attach to this
material. the indirect contact testing involves the observation of these
cells in the well. When toxic compounds are leached, the cells may be
(in)directly affected and will show reduction in adhesion and growth.
At different time points (e.g. 24h, 48h) the cells are made visible on a
microscope, compared to a control group. You may see differences in the cells, like not a whole
surface covered in cells, but this does not always mean that it is due to cytotoxicity (could also be a
surface that they do not like during direct contact & they will then not adhere). Surface properties
play an important role on how cells behave. To see whether is was due to the surface properties or
cytotoxicity, you can test the same materials and cells with extract testing. You can then see if the
extraction gives rise to MTT metabolic activity (if not → cytotoxicity). So to sum up:
Unlikely cell behavior during direct contact testing → use extract testing → according to extract
testing the cells
- Are lively / proliferation well → the surface properties were not right during contact testing.
- Not lively / not proliferating well → there are cytotoxic leachables in the tested material.
It can be a combination of both: not a good surface to adhere to & cytotoxicity.
Passivation = an intermediate in the coating that is potentially toxic & when it is not covered /
enclosed by anything else, it can leach. E.g. an Fn coat can stop leaching from a linking polymer in a
