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Biocompatibility testing and marketing authorisation of degradable magnesium implants 341
Biological safety
evaluation of implants
Parasites, bacteria and
fungi
ISO standards for
sterilisation
prions and other
Materials of animal
origin:
ISO 22442 series
Virus,
transmittable
pathogens
Viable human cells:
ISO 13022
Organic or inorganic
leachables
contaminations or
components
ISO 10993 series
Figure 11.5 Biological safety evaluation of implants involves the assessment of the risk
of pathogens and chemical contaminations (leachables). Various ISO standards give guidance
on risk evaluation and minimisation.
Implants should also be free of transmittable pathogens such as viruses or prions.
Contamination with these pathogens may occur if they have been made with or consist
of materials of animal origin, e.g., bovine serum albumin, collagen, trypsin or pepsin,
amino acids, collagen, or deoxycholate. Furthermore, human cells can also transmit
pathogens if they are part of the implant. A strategy of how to evaluate and control
the risk of infection with respect to materials of animal origin or human cells is
described in the ISO standard series 22442 or standard 13022 (Figure 11.5). Biological
materials are difficult and sometimes impossible to sterilise with conventional methods
using heat or radiation because they become inactivated or are destroyed during the
process. In these cases, it is common practice to work under sterile conditions. Guidance concerning this topic will be given by new ISO standard 18362 (Table 11.2),
which will be published probably in 2015.
In addition to biological contamination, the chemical composition of the starting
material, production residues (e.g., organic solvents, separating agents), leachables,
degradation products, wear debris, ageing, or surface effects have to be taken into account for the biological evaluation of implants. The material-related aspects are tested
and evaluated according to the ISO 10993 series (Figure 11.5). The ISO 10993 series
consists of 20 parts, and a new section (Part 22) for nanomaterials is currently under
development (Table 11.3). The set of tests that should be considered for evaluating
the safety of a medical device depends on the manner and duration of contact with
the body. A re-evaluation is necessary for the following types of changes: a change
of supplier for the materials; specification changes; an alteration of the composition,
processing, packaging, or sterilisation process; or if the material is used for a new application. Other reasons for re-evaluation could be the indication of adverse effects on
humans after the product is on the market or if the product is altered during storage.
342 Surface Modification of Magnesium and its Alloys for Biomedical Applications
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Table 11.2 Exemplary list of standards that cover the topic of
sterilisation of medical devices
Topic Number Title
General requirements EN 556-1 Sterilisation of medical
devices e Requirements
for medical devices to be
designated “STERILE,”
part 1: Requirements for
terminally sterilised
medical devices
EN 556-2 Sterilisation of medical
devices e Requirements
for medical devices to be
designated “STERILE,”
part 2: Requirements for
aseptically processed
medical devices
ISO 17664 Sterilisation of medical
devices e Information to
be provided by the
manufacturer for the
processing of
resterilisable medical
devices
Technique & equipment EN 285 Sterilisation e Steam
sterilisers, large
sterilisers
EN 13060 Small steam sterilisers
EN 1422 Sterilisers for medical
purposes e Ethylene
oxide sterilisers e
Requirements and test
methods
EN 14180 Sterilisers for medical
purposes e Lowtemperature steam and
formaldehyde sterilisers
e Requirements and
testing
Biocompatibility testing and marketing authorisation of degradable magnesium implants 343
Table 11.2 Exemplary list of standards that cover the topic of
sterilisation of medical devices e cont'd
Topic Number Title
Methods EN ISO 11135-1 Sterilisation of health care
products e Ethylene
oxide, part 1:
Requirements for
development, validation,
and routine control of a
sterilisation process for
medical devices
EN ISO 11137-1 Sterilisation of health care
products e Radiation,
part 1: Requirements for
development, validation,
and routine control of a
sterilisation process of
medical devices
EN ISO 11137-2 Sterilisation of health care
products e Radiation,
part 2: Establishing the
sterilisation dose
EN 13824 Sterilisation of medical
devices e Aseptic
processing of liquid
medical devices e
Requirements
EN ISO 14160 Sterilisation of single-use
medical devices
incorporating materials
of animal origin e
Validation and routine
control of sterilisation by
liquid chemical sterilants
EN ISO 14937 Sterilisation of health care
products e General
requirements for
characterisation of a
sterilising agent and the
development, validation,
and routine control of a
sterilisation process for
medical devices
Continued
344 Surface Modification of Magnesium and its Alloys for Biomedical Applications
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Table 11.2 Exemplary list of standards that cover the topic of
sterilisation of medical devices e cont'd
Topic Number Title
EN 15424 Sterilisation of medical
devices e Lowtemperature steam and
formaldehydee
Requirements for
development, validation
and routine control of a
sterilisation process for
medical devices
EN ISO 17665-1 Sterilisation of health care
products e Moist heat,
part 1: Requirements for
the development,
validation, and routine
control of a sterilisation
process for medical
devices
ISO 18362 Processing of cell-based
health care products
The set of tests described in the ISO 10993 series are often referred to as “biocompatibility testing.” Alternatively, if an implant passes all necessary tests mentioned in
ISO 10993, it is described as “biocompatible.” The word comes from the Greek bios
(life) and compatible (compliant). The term biocompatibility was first defined by David F. Williams in the late 1980s as “the ability of a material to perform with an appropriate host response in a specific application” (Williams, 1987). In 2008, he redefined
the earlier version of his definition as follows: “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 effects in the recipient or beneficiary of that
therapy, but generating the most appropriate beneficial cellular or tissue response in
that specific situation, and optimising the clinically relevant performance of that therapy” (Williams, 2008). Later, the International Union of Pure and Applied Chemistry
(IUPAC, www.iupac.org)defined biocompatibility as the “ability of a material to
perform with an appropriate host response in a specific application” and defined
biomedical therapy as the “ability to be in contact with a living system without producing an adverse effect” (Vert et al., 2012 ).
Biocompatible materials can also be broken down by type as “bioinert,”
“biotolerant,” or “bioactive.” A bioinert material shows virtually no chemical or biological interaction with the surrounding tissue, whereas a bioactive material interacts with
Biocompatibility testing and marketing authorisation of degradable magnesium implants 345
Table 11.3 List of all currently available parts of ISO 10993 “ Biologic
evaluation of medical devices”
10993-1 Evaluation and testing
10993-2 Animal welfare requirements
10993-3 Test for genotoxicitiy, carcinogenicity and reproductive toxicity
10993-4 Selection of tests for interactions with blood
10993-5 Test for in vitro cytotoxicity
10993-6 Test for local effects after implantation
10993-7 Ethylene oxide sterilisation residuals
10993-8 Selection and qualification of reference materials for biological tests
10993-9 Framework for identification and quantification of potential degradation
products
10993-10 Test for irritation and delayed-type hypersensitivity
10993-11 Test for systemic toxicity
10993-12 Sample preparation and reference materials
10993-13 Identification and quantification of degradation products from polymeric
medical devices
10993-14 Identification and quantification of degradation products from ceramics
10993-15 Identification and quantification of degradation products from metals and
alloys
10993-16 Toxicokinetic study design for degradation products and leachables
10993-17 Establishment of allowable limits for leachable substances
10993-18 Chemical characterisation of materials
TS 10993-19 Physicochemical, morphological and topographical characterisation of
materials
TS 10993-20 Principles and methods for immunotoxicology testing of medical devices
10993-22 Guidance on nanomaterials (in development)
Parts of ISO 10993 that include test procedures using implant extracts are in italics
the surrounding tissue and is typically used where a tight connection between tissue and
implant material is required. In contrast, a biotolerant material is not bioactive and, in
the long run, not bioinert either. However, it can be safely used for a certain time
(from several months to several years) before compatibility with the surrounding tissue
starts to fade/diminish. Interestingly, the term biocompatibility or biocompatible is not
defined in the European or American laws on the requirements and procedures of marketing authorisation. It is also not defined in any of the ISO standards mentioned above.
346 Surface Modification of Magnesium and its Alloys for Biomedical Applications
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In summary, the term “biocompatibility” has various definitions. In the context of
marketing authorisation, it refers to the property of an implant (medical device) that is
mainly confirmed by passing a set of tests documented in ISO 10993. Biocompatibility
is only one aspect of the biological evaluation of implants.
11.5 Preclinical biocompatibility testing
of biodegradable magnesium implants
for marketing authorisation
11.5.1 General principles of biocompatibility testing
for marketing authorisation
The aim of biocompatibility testing for marketing authorisation is to demonstrate that
the implant is safe for its intended use (“fit for purpose”), which is determined by the
manufacturer. To make sure that the implant is fit for purpose, all potential dangers
should be taken into account; however, they do not necessarily need to be tested. It
is also possible to replace testing by an evaluation of the relevant literature, especially
to avoid unnecessary animal experiments. All of the available literature can be used for
the evaluation as long as the evalua tion process is explained in a comprehensive
manner so that the regulatory bodies can follow the argumentation easily and are
able to evaluate the suitability of the implant for its intended use.
In vitro and in vivo testing should always be conducted with the final product (or a
test item that has been treated like the final product), which means that it has to already
be packaged and sterilised (original packaging). This is done to ensure that no changes
have occurred to the imp lant and that toxic substances have not been released from the
packaging and come into contact with the implant during the sterilisation process.
Given that ageing can also induce changes to the sterilised implant in its primary packaging, tests of the final aged product should be considered. The extent to which the
final aged product needs to be tested has to be evaluated by risk management.
The test items to be used for biocompatibility testing should be traceable and
marked accordingly (e.g., with their designations and batch numbers). Tests for marketing authorisation should be performed by certified laboratories (EU: ISO 17025,
USA: Good Laboratory Practice, GLP). Some companies do the preclinical testing
in-house; in that case, their laboratories will be included in the product audit for
approval by the regulatory authorities or regulatory competent bodies. In the interest
of reducing animal testing, animal experiments performed at universities or other
noncertified laboratories can also be used for marketing authorisation if they meet
the ISO 10993 requirements. Currently, there is discussion about whether these data
should be excluded from studies for marketing authorisation; therefore, it might be
possible that only data collected by certified laboratories are accepted by the regulatory
bodies in the future. In any case, all the test results have to be documented in a test
report, including the raw data. Finally, the manufacturer has to summarise its conclusions based on the tests performed.
Biocompatibility testing and marketing authorisation of degradable magnesium implants 347
11.5.2 Standards for biocompatibility testing of magnesium
implants
Currently, there are no specific standards for testing the biocompatibility of biodegradable magnesium implants. However, some efforts have been made to adapt ISO 10993
“biological evaluation of medical devices” to issues related to degradable (magnesium)
implants. As a result of these efforts, TR 37137, titled “Biological evaluation of medical devices Guidance for absorbable implants,” has been published recently. The
document is based on a gap analysis and lists changes to ISO 10993, which are required
for biocompatibility testing of absorbable implants made of polymers or metals. The
contents of the document will be discussed within the working groups of the ISO technical committee 194 “Biological and clinical evaluation of medical devices,” which develops the 10993 series. If the experts within the working groups agree to the proposed
changes, they will be included gradually in the next revision. Complementary to TR
37137, the ASTM document F1983-99 (2008) “Standard Practice for Assessment of
Compatibility of Absorbable/Resorbable Biomaterials for Implant Applications” gives
guidance on the examination of tissue reactions to absorbable biomaterials for implants
that will reside in bone or soft tissue longer than 30 days and less than 3 years.
There are also few application-specific standards available. On the international
level, ancillary to ASTM F3036-13 “Standard Guide for Testing Absorbable Stents,”
an ISO document dealing with absorbable cardiovascular implants either based on
polymers or metals (ISO/TS 17137) has been recently published. For aspects related
to biocompatibility, this document refers to ISO 10993. Also an ASTM standard is
available that gives guidance to mechanical testing of orthopaedic absorbable implants
(plates and screws for internal fixation, ASTM F2502-11).
11.5.3 Biocompatibility testing of implants according to ISO
10993
ISO 10993, “Biological evaluation of medical devices,” is a semihorizontal level 2
standard that consists of 20 parts (Table 11.3) and is applicable to all kinds of medical
devices as well as implants. Examples of medical devices are hospital bed linens, plaster, wound dressing materials, ultrasound gels, dialysis equipment, surgical inst ruments, condoms, contact lenses, catheters, artificial hips, and stents. Part 1 of ISO
10993 provides guidance on the biological evaluation and use of the standard. Besides
biological tests, several chemical analyses of degradation products and leachables,
which are also are part of the biocompatibility assessment of a product, have to be
considered (Figure 11.6). Annex A in ISO 10993-1 gives an overview of the tests
required for biocompatibility assessment. Since the standard is relevant for many
different products, the manner and duration of body contact of a medical device is a
decisive factor for the toxicological topics, which has to be addressed in the biocompatibility evaluation either as a test result or in relation to the existing literature. For
implants, usually all tests mentioned in ISO 10993 have to be taken into account.
The only exception applies to implants that are not exposed to blood, in which case
the tests for hemocompatibility (ISO 10993-4) may be omitted.
348 Surface Modification of Magnesium and its Alloys for Biomedical Applications
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Chemical
characterisation
ISO 10993–18
Determination of limits
ISO 10993–17
No
Identical to licensed
product
No
Determination of the
chemical composition
Toxic potential?
Literature!
Yes
Toxicological assessment
ISO 10993–16, in vivo
Well-established
limits?
Yes
Leachables?
ISO 10993–9, 10993–15,
literature
Yes
No
Yes
No
Biological testing ISO 10993–3, –4, –5, –6, –10, –11
Figure 11.6 Biocompatibility evaluation of medical devices adapted from ISO 10993-1.
11.5.4 Specific considerations for testing magnesium-based
implants
Due to the general character of ISO 1099 3 , not all medical devices can be tested in
accordance with this standard without encountering some difficulties. In the case of
degradable magnesium implants, their abil ity to dissolve causes problems when
some parts of ISO 10993 are applied. This is because many of the tests require the
preparation of an aqueous extract of the implant, for example “Genotoxicity” (Part
3), “Hemocompatibility” (Part 4), “Cytotoxicity” (Part 5), “Irritation, Sensitization”
(Part 10), and “Systemic toxicity” (Part 11). If a magnesium implant is extracted in
an aqueous solution, it degrades to magnesium, hy droxide ions, and hydrogen gas.
The degree of degradation is reflected in the increase in osmolality and the pH of
the extract, both of which can also lead to positive results for toxicity. The in vitro
degradation of magnesium alloys is enhanced compared to the in vivo situation
(Bobe et al., 2013; Witte et al., 2006) depending on the extraction media (Bobe
et al., 2013; Feyerabend et al., 2012; Willumeit et al., 2011; Xin, Hu, Chu, 2011).
This has to be taken into account for evaluation of the results from the extractionbased testing methods according to ISO 10993. In cases of a very fast in vitro degradation of the magnesium implant, the tests have to be adapted or they might not be
suitable for biological evaluation. Besides the extraction-based tests (originally
designed to detect toxic production residues, leachables, and degradation products
of permanent polymer or metallic implants), animal experiments can also lead to false
results if unsuitable test items, implantation sites, or animal models are chosen. Since
vascularisation, gas, and fluid exchange are factors that play an important role in in
Biocompatibility testing and marketing authorisation of degradable magnesium implants 349
vivo degrada tion (reviewed in Witte (2010)), it is strongly advisable to choose the
implantation site according to the intended use of the implant.
11.5.4.1 Necessary modifications to biological tests of ISO 10993
for magnesium-based implants
For the extraction-based tests (Parts 3, 4, 5, 10, and 11), it is recommended that cell
culture media or a medium similar to body fluids (Xin et al., 2011; Willumeit et al.,
2011; Feyerabend et al., 2012) be used for extraction and that the extraction be
performed at a temperature of 37
(ISO 10993 Parts 3, 4, and 5), it is generally a good idea to test the final product at
various stages of degradation to check for the toxicity of by-products. The osmolality
and pH of the extract should be controlled to avoid misinterpretation of the test results.
However, ISO 10993-12:2012, “Sample preparation, controls and reference materials,” states that the pH of the extract “shall not be adjusted unless a rationale is provided.” Adjusting the pH for extraction-based indirect cytotoxicity testing according to
ISO 10993 Part 5 can be justified given that basic pH is cy totoxic itself and the increase
in pH might interfere with test components, e.g., XTT, MTT, or MTS (Fischer et al.,
2010). High pH values, high osmolality, and a high cytotoxic potential of the product
extract could also affect genotoxicity testing. In addition, testing of various extract dilutions might also be necessary.
For in vivo experiments, a pH adjustment could be necessary to a void misleading
results if the implantation site does not reflect the actual in vivo cell exposure conditions (e.g., in terms of physiological perfusion) (Hoffheinz and Dimitroff, 1928)
in irritation tests (ISO 10993 Part 10) or tests for systemic toxicity (Part 11). If
an adverse pathology is monitored, an assessment of the reversibility of the
observed effects after a ce rtain time should be considered. During implant retrieval
and tissue sample collection, care should be taken to minimise artefacts due to
histological processing and handling (e.g., further degradation of the material or
shrinkage).
The development of hydrogen gas can be problematic in in vivo tests. The misinterpretation of in vivo results can be reduced by selecting an implantation site according to the intended use of the implant.
Cellular attachment to fast degrading magnesium implants might not be possible to
be studied in vitro, because the fast changes to the surface resulting from the release of
hydrogen gas prevent cellular attachment. However, this functional test is not part of
ISO 10993; therefore, it is not relevant for marketing authorisation.
C(Xin et al., 2011). With regard to the in vitro tests
11.5.4.2 Additional options for the modification of standard
protocols in ISO 10993-5
A simple way to avoid adjustment of the pH could be to use a test system not affected
by a high concentration of hydroxide ions (e.g., neutral red staining). Some other
alternatives could be counting the cell number, estimating the total protein, or using
a qualitative live/dead assay based on cell membrane integrity. Fischer et al. (2010)
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proposed using a BrdU (5-bromo-20-deoxyuridine) incorporation assay, which measures cell proliferation and is not disturbed by the pH. However, the latter is not advisable for cytotoxicity testing for marketing authorisation, because according to ISO
10993-5 the output of the test has to demonstrate cell viability. Cell proliferation
can contribute to viability; however, nonproliferating cells can also be viable. XTT,
MTT, or MTS tests can also be applied with a slight modification of the protocol:
One example is to exchange the cell culture medium prior to XTT, MTT, or MTS
application (Witte et al., 2006). Another possibility is to perform the test in parallel
without cells to determine the absorption baseline. The latter can be further subtracted
from the measured values to obtain the corrected baseline values (Fischer et al., 2010).
In addition to the problem of pH and osmolality changes, the released magnesium ions
(or other ions) might also affect the test by interacting with test components. Therefore,
compatibility of the magnesium alloy with the test method should be tested before.
Since the composition of the extraction media has a huge impact on the degradation
of magnesium alloys and ISO 10993 does not specify the use of a specific medium, the
choice of cell culture medium might also be worth considering. Commonly used cell
lines for cytotoxicity testing, such as L929 or NIH 3T3 fibroblasts, are relatively undemanding and grow in many standard cell culture media such as RPMI 1640,
DMEM, or MEM. All of the media mentioned are commercially available with
different compositions with regard to their glucose, salt, and buffer concentration.
Some companies are also willing to produce individualised cell culture media according to the needs of the customer.
Another factor to consider is the cell type used for the in vitro experiments. According to ISO 10993 Part 5, several cell lines are proposed for cytotoxicity testing, and it is
possible to use primary human cells as well. Current publications indicate that the results of cytotoxicity testing might differ depending on the cell line used (e.g., Bobe
et al., 2013; Feyerabend et al., 2010). Therefore, it might support the biological eval-
uation to use a cell type that reflects the actual in vivo cell exposure.
To summarise, the aim of biocompatibility testing for marketing authorisation is to
demonstrate that the implant is safe for its intended use (“fit for purpose”), which is
determined by the manufacturer. Currently, there are no specific standards for testing
the biocompatibility of biodegradable magnesium implants. The standards of the ISO
10993 series can be applied, but the protocols might need slight modifications and
additional controls, e.g., pH and osmolality measurements. Adaption of the ISO
10993 series according to the requireme nts of degradable magnesium implants will
be pursued in a stepwise manner during the periodic revisions of the documents.
11.6 Nonstandardised in vitro and in vivo techniques for
biocompatibility evaluation of magnesium alloys
A survey of the available literature demonstrates that most of the groups use in vitro
viability tests such as MTT or XTT for biocompatibility assessment. Some groups
have also published results using neutral red live/dead assays (two fluorescent
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