Evaluation of Biocompatibility Using In Vitro Methods: Interpretation and Limitations - Tissue Engineering: Cell-Surface Interactions for Tissue Culture - page 136

3.1 General Limitations of In Vitro Bioactivity Measurements

The advantage of in vitro systems is that they are simple and that all parameters
can be defined. Furthermore they are cheaper and less time-consuming than in vivo
tests. However, current in vitro systems also have their drawbacks:
1. Cell source. As for cytotoxicity tests, cell line cells used for these studies are
often of animal, not human, origin (e.g. mouse MC3T3 or human MG63 cells
for osteoblasts [ 46 , 74 , 105 , 119 ].
2. Assumptions. The assessments are based on certain assumptions which are often
not correct, since the prognostic values of in vitro bioactivity parameters versus
in vivo performance have not been systematically evaluated. For example, the
assumed correlations between in vivo performance and the degree of cell
adherence, cell proliferation rate and cell differentiation are not valid (e.g. a
parameter of early osteoblast differentiation, alkaline phosphatase (ALP)

activity, is increased [ 74 ]).
3. Heat-inactivated serum. Heat inactivation alters the conformational state of
heat-labile proteins. It is known that heat inactivation of these proteins may
affect cell functionality and cell-surface interactions [ 25 , 37 ]. Furthermore,
culture medium containing a dilution of heat-inactivated fetal calf serum may
give a result for a protein layer not comparable to one present after implantation
in an environment of 100% interstitial fluid or serum, both of which are not
heat-inactivated [ 97 ]. As a result, not only cell spreading but also proliferation
and differentiation may be affected. The use of media with denatured proteins
thus certainly represents one important drawback of current in vitro systems for
selecting biomaterials, investigating cell material interactions and elucidating
the underlying mechanisms.

In most studies in which the prognostic value of in vitro studies is a theme, a
comparison of these studies with animal studies is made. However, since the
prognostic value of animal studies for the human situation is also limited, the
outcome of such a comparison is questionable and it might be discussed how far
such a comparison should be made at all.

3.1.1 Example: Cell Adherence

Generally, it can be assumed that cell adherence is directly related to the cell's
ability to interact with the surface. Experimentally, cells are seeded on top of the
samples and cell adherence is measured by quantifying cell number at defined
times thereafter. The incubation period used varies depending on the research
group: 1 h [ 105 ],2h[ 99 ], 3 h [ 74 ], 4 h [ 81 , 117 ], 5 h [ 106 ], 0.5-6 h [ 140 ]or24h
[ 85 ]. However, comparing in vitro with in vivo data, such a correlation could
not always been found (a correlation is seen in, e.g. [ 81 , 99 ], but not in, e.g.
[ 105 , 117 ]). One explanation could be that cell adherence is measured before