Evaluation of Biocompatibility Using In Vitro Methods: Interpretation and Limitations - Tissue Engineering: Cell-Surface Interactions for Tissue Culture - page 126

2.2.1 Limitations of Extraction Procedure
Extraction of compounds from materials is not as simple as it sounds. The stan-
dard describes different extraction fluids for preparing extracts, and each one has
its own limitation. Generally speaking, the proposed fluids are not designed to
extract compounds from a material! It is a very mild procedure which detaches
some compounds from the surface and may extract very soluble ones from the
near surface bulk material, but the procedure does not reflect in vivo conditions in
any way, i.e. prolonged contact between cells/tissue/tissue fluids and the material.
The extraction yield can be easily \10%, depending on the extractable com-
pounds, as compared to an exhaustive extraction. In particular, the approach is
not suited for polymeric systems that may contain extractable hydrophobic
constituents, as, for example, is well-known from total organic carbon (TOC)
measurements [ 87 ].
The standard suggests three extraction fluids: (a) culture medium with serum,
(b) physiological saline buffer, or (c) other ''suitable extraction vehicles'' including
pure water or dimethyl sulfoxide (DMSO) \0.5%. The use of culture medium
containing serum has the disadvantage that the material surface is immediately
covered by a protein layer of albumin and fibronectin. This layer passivates the
material interface, limiting the release of possible toxic components. Here it must
be noted, however, that this kind of barrier is also formed after implantation of the
material and thus in principle mimics the in vivo situation to a certain extent. The
use of physiological saline buffer is only suited for the extraction of hydrophilic
compounds including soluble salts. Other extraction fluids such as pure water or
fluids containing minute amounts of DMSO allow for comprehensive extraction of
neither hydrophobic and hydrophilic components. Furthermore, DMSO (1-2%) is
known to alter the cell phenotype [ 51 , 131 ]. However, the goal and at the same
time also the problem of the extraction procedure is that it should capture all
compounds that might be released from the surface or that might diffuse out of the
material during the lifetime of the device—which might be many years. Obvi-
ously, hydrophilic soluble substances are not seen as a problem for collection in
the extract, but most organic hydrophobic substances will escape the collection
since their saturation in an aqueous medium is extremely low. Furthermore, dif-
fusion processes in polymers are usually very slow and apolar solvents are
required to speed the process up.
The ISO 10993-5 standard also dictates the extraction temperature and time.
Four different standard conditions are given: 24 h at 37C, 72 h at 50C, 24 h at
70C, and 1 h at 121C. Among the different extraction temperatures suggested,
37C is certainly a physiological temperature and matches cell culture and serum
extraction media. The advantage of using higher temperatures (50/70/121C) is
that the release of components may be induced that would not be released under
normal conditions. However, this may occur with the disadvantage that the
released components may be modified due to the higher temperature. Therefore, by
using higher extraction temperatures the absence of these modifications needs to
be ensured to prevent the occurrence of false positives and negatives.