Bioresorption and Degradation of Biomaterials - Tissue Engineering: Cell-Surface Interactions for Tissue Culture - page 325

3.2 Cell-Based Resorption Assays
For reasons of standardization, ISO testing of material degradation disregards
many of the influences observed in vivo, such as active cellular influences. This
may be an explanation why some materials which were shown to be fully
degradable under ISO conditions have still been detected after years of in vivo
implantation [ 86 , 87 ]. Therefore, to improve the prediction of in vivo behavior,
cell-based degradation testing has been developed (Fig. 1 ).
Several kinds of cells have the ability to resorb foreign materials such as
osteoclasts and macrophages [ 88 , 89 ]. The most commonly used cells for ana-
lyzing resorption in vitro are osteoclasts. This is probably due to the widespread
clinical application of such materials in both dental and orthopedic patients.
Primary osteoclasts are found in abundance in the bones of neonatal animals
[ 90 ]. In 1984, Chambers and colleagues isolated osteoclasts from neonatal rabbit
long bones by fragmenting the bone structure in HEPES-buffered medium [ 91 ].
Similar isolation procedures were then transferred to neonatal rat and neonatal
chicken [ 92 ]. In addition, Jones et al. [ 93 ] found that osteoclasts isolated by the
same approach showed no differences in species (rat, chicken or rabbit) with
regard to biomaterial resorption. This method was considered by some
researchers to be the best for the isolation of large numbers of primary osteo-
clasts [ 94 ]. Besides primary osteoclasts, differentiated osteoclasts, especially
differentiated human osteoclasts, have recently been studied for this purpose,
because they promise to permit individualized testing of the material with the
cells of specific patients [ 52 , 53 ]. Two factors are essential for osteoclast dif-
ferentiation: receptor activator of nuclear factor kappa-B ligand (RANKL) and
macrophage colony-stimulating factor (M-CSF). Both RANKL and M-CSF are
secreted by osteoblasts for activation of osteoclast differentiation. M-CSF is a
hematopoietic growth factor involved in the proliferation and differentiation of
monocytes [ 95 ]. Although indispensable for osteoclast differentiation, high
concentrations of M-CSF actually inhibit osteoclast formation in vitro. The
identification of RANKL as a differentiation factor for osteoclasts and its
recombinant production has had a major impact on cell-based resorption assays
[ 96 ]. Isolation of mononuclear precursors from different sources and species and
differentiation into osteoclasts has since become more and more popular in cell-
based resorption assays.
Since cellular resorption typically leads to only subtle changes in the surface
structure of the material, the evaluation of the material mass is usually not suitable
for cell-based resorption assays and specialized methodology is necessary.
The analysis of surface resorption of biomaterials is commonly based on
several classical and also advanced optical methods such as light microscopy
(LM), scanning electron microscopy (SEM), confocal laser scanning microscopy
(CLSM) and infinite focus microscopy (IFM). For two-dimensional (2D) analysis,
LM is still the first choice. This is partly due to the wide availability of such
equipment and also the ease of the procedures. However, the LM resorption assay