Influence of the Processes Parameters on the Properties of The ...

Chapter 1.
Polylactide Based Bio-Materials
that secretion <strong>of</strong> some specific growth factors is up-regulated. O<strong>the</strong>r requirements are a good porosity and
<strong>the</strong> need <strong>of</strong> bioreactors and gas exchanges during culture. Tumour cells may also be implanted in <strong>the</strong>
scaffold subcutaneously in <strong>the</strong> back <strong>of</strong> mice. This method is limited because no up-scaling <strong>of</strong> this procedure
is possible and no observation is possible during culture. These models seem to be restricted to research
laboratories.
1.1.2.4 Cells Grown in a Biomaterial at a Micrometer-Scale (Thickness ~200 µm)
This material has its limitation because cells may behave differently depending on <strong>the</strong> batch <strong>of</strong>
reconstituted basement membrane extract (BME) called Matrigel ® , <strong>the</strong>y are grown on. Because BME is a
natural product made from extracts <strong>of</strong> mouse sarcomas, it is difficult to get uniform, consistent preparations,
even from <strong>the</strong> same manufacturer. The solution is to test and to stock validated batches <strong>of</strong> BME. There are
several o<strong>the</strong>r methods such as matrix production by fibroblasts or mixture <strong>of</strong> tumour cells and matrix. For
<strong>the</strong> former, alkaline treatment removes cells that leave behind complex substrates <strong>of</strong> fibronectin, collagen,
and o<strong>the</strong>r proteins. Progression <strong>of</strong> <strong>the</strong> tumours deriving from <strong>the</strong>se matrices laid down normal fibroblasts
[Fischbach et al., 2007]. For <strong>the</strong> latter, a gel mixture is prepared by mixing an extracellular matrix (ECM)
with tumour cells and Matrigel ® (volume to volume 1:1) that allows <strong>the</strong> solution to polymerize. The
resulting 3D scaffold on which tumour cells are cultured is not <strong>the</strong> pure ECM, <strong>the</strong>y would encounter in vivo.
Ano<strong>the</strong>r alternative is to use commercial 3D matrices. These are, for example, reconstituted
basement membrane extract such as Matrigel ® (Sigma). Matrigel ® is used to support growth and
differentiation <strong>of</strong> cells and tissues. It recapitulates <strong>the</strong> morphology and visco-elasticity <strong>of</strong> <strong>the</strong> extracellular
matrix and can be remodelled by cells. The drawbacks are that it is expensive and <strong>the</strong> composition is
variable. Alternatively, interstitial matrix components such as collagen, fibrin, etc may be used. They are
frequently employed to study migration and invasion. These matrix proteins can be remodelled by cells, and
are <strong>of</strong>ten used in combination. However, <strong>the</strong>ir use may be problematic because glycosylation and solubility
may vary by source and <strong>the</strong> properties between native and denatured proteins may differ. There are o<strong>the</strong>r
commercially available matrices such as (semi)-syn<strong>the</strong>tic hydrogels proposed by BD (PuraMatrix peptide
hydrogel). It <strong>of</strong>ten polymerizes in combination with bioactive peptides. The drawbacks are that it shows
some bioactivity with certain cells and it cannot be remodelled or degraded by cells. O<strong>the</strong>r ready-to-use
systems are <strong>the</strong> algiMatrix3D ® culture system (invitrogen) that comes as sponges in 96-well plates or
InsertTM-PCL (3D Biotek ® ), a biodegradable polycaprolactone scaffold. These systems are quite
expensive. O<strong>the</strong>r authors Castelló-Cros and Cukierman [2009] have used 3D agarose colony formation and
GelCount technology with GelCount scan and image acquisition for high-resolution scanner allowing
<strong>the</strong> calculation <strong>of</strong> half maximal inhibitory concentration (IC50). This system provides quantitative data (log
dose and time-dependent effects <strong>of</strong> drugs). However, no description <strong>of</strong> <strong>the</strong> gel (thickness) and <strong>of</strong> <strong>the</strong> quality
<strong>of</strong> colonies formed is reported for this model (size <strong>of</strong> <strong>the</strong> colony, interactions between cells, and <strong>the</strong>
distribution <strong>of</strong> <strong>the</strong> cells across <strong>the</strong> colony).
1.2 Bio-Composites for Calcified Tissue Engineering
1.2.1 Composition <strong>of</strong> Scaffolds
The principal calcified tissue <strong>of</strong> vertebrates is bone. O<strong>the</strong>r calcified tissues in vertebrates include
calcified cartilage, which is present to some extent in most bones and <strong>the</strong> dental tissues - enamel, cementum,
and dentin. Bone develops by <strong>the</strong> process <strong>of</strong> ossification, osteogenesis, as a specialized connective tissue.
During ossification, osteoblasts secrete an amorphous material, gradually becoming densely fibrous -
osteoid. Calcium phosphate crystals are deposited in <strong>the</strong> osteoid (i.e. mineralization), <strong>the</strong>reby becoming bone
matrix. Osteoblasts become surrounded during <strong>the</strong> mineralization process, and <strong>the</strong> cells become osteocytes.
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