Evaluation of Biocompatibility Using In Vitro Methods: Interpretation and Limitations - Tissue Engineering: Cell-Surface Interactions for Tissue Culture - page 141

proteins to withstand the toxic insults of hypoxia, and releasing vasoactive
factors to re-establish proper perfusion and thus oxygen supply. Under these
conditions, chondrocytes up-regulate collagen II production and aggrecan pro-
duction [ 53 ]. An elegant approach to mimicking the in vivo oxygen tension
gradient as well as an ECM environment is to place a collagen or agar gel layer
with embedded cells on top of the test material. With this set-up, another
important in vivo aspect would be taken into consideration—the dimensionality
(3D instead of 2D). Collagen gels with embedded cells are currently used for
various investigations including gene transfer [ 61 ], effects of matrix stiffness
[ 63 ], mechanical load and tissue engineering [ 145 ]).
All these are single individual attempts to improve the test system by taking at
least one key issue of the in vivo niche in the direct vicinity of the implant into
account. However, a combination of several of these modifications of the test
system would certainly help to better mimic this niche and as a result to further
improve the test system.
4.1 Proposed Testing Strategy
By assessing in vitro biocompatibility of new materials in principle, answers to ten
important questions are sought. In Fig. 6 these questions are listed together with
which kind of tests we think could be used to find an answer. Since current testing
strategies for biocompatibility have strong limitations regarding their prognostic
value, we propose a multi-level sequential approach in which cytotoxicity is
evaluated in the first of two levels. The first two levels are similar but not identical
to the ISO 10993-5 test. In subsequent tests, bioactivity is investigated. For this, no
in vitro test standards are currently present. The proposed multi-level test sequence
is shown in Fig. 7 . The complexity of the tests and their specificity increases from
one level to the next. Each level focuses on another aspect relevant for biocom-
patibility and the biological performance of a material. In total, this test sequence
should give an answer to all the questions mentioned in Fig. 6 . The set-up pre-
sented assumes that the material is implanted for at least five days. The cells used
for the first two levels are based on cell lines that are representative for the site of
implantation of the material. Subsequent levels are based on primary cells (opti-
mally human cells), again specific for the site of implantation. The various levels
can be described as follows:
• Level 1—direct contact test (induction of cell necrosis): The first level is
identical to the ISO 10993-5 direct contact test. With this test, a statement on the
induction of acute cell necrosis can be made (within 24 h) (=[Answer to
question 1).
• Level 2—extract test (effects on cells mass): For the second level, slight
modifications are suggested to the ISO 10993-5 extract test. Instead of sub-
confluent cultures to which the extract is given, a seeding density is chosen