CHITIN AND CHITOSAN FOR TISSUE ENGINEERING APPLICATION - Intelligent Scaffolds for Tissue Engineering and Regenerative Medicine - page 443

protein deposition resulting in the formation of bigger aggregates
compared with conventional pellet culture. 28 Park et al. reported
a double-bead microsphere device to deliver bioactive molecules
to cells. These double-beaded PLGA microsphere constructs con-
tainingdexamethasoneanddehydroepiandrosterone show promise
as coatings for implantable biomedical devices to improve biocom-
patibility and ensure in vivo performance. 29 Park and Kim also
developed chitosan bead scaffolds having interconnective micro-
pores between macropores by the modified TIPS process. 7 This
method isdetailed below.
23.4.1 Preparation of Chitosan Bead Scaffolds
Chitosan (75.6% deacetylated) was dissolved in 1% acetic acid
to give 1% and 2% (w/v) solution. Ten milliliters of 20% (v/v)
n-butanol/1% acetic acid solution was dropped into 10 mL of 2%
chitosan solution. Final volumes of n-butanol were 10% in the
1% chitosan solution. The solution was homogenized to obtain the
n-butanol/chitosanmixtureandwasthenpouredinto5mLsyringes
with needles. The 18G and 23G needles were used for the 10%
n-butanolsolutionandthe1%chitosansolution,respectively.Drops
of the chitosan solution were extruded manually from the syringes
into a beaker containing a cooling medium. Methylene chloride
(MC), an MC/ether mixture, and liquid nitrogen (LN 2 ) were used
as the cooling media. The temperature of the cooling medium was
controlled at -15 ◦ C, -30 ◦ C, and -70 ◦ C by a refrigerator, or into LN 2
(-196 ◦ C) directly. After five minutes for LN 2 and three hours for
other cooling media, the completely solidified chitosan beads were
moved to a cooled, freeze-dried glass vessel for freeze-drying. The
solidified chitosan beads were freeze-dried at -70 ◦ C for six hours.
The density of the cooling medium at the set temperature should
be only slightly less than that of the chitosan solution. Therefore, a
32%MC(inether)solutionwasusedasthecoolingmediumfor10%
n-butanol(in 1% chitosan) solution at -15 ◦ C, -30 ◦ C, and -70 ◦ C.
To fabricate beads with a larger pore size, the cooling rate was
controlledslowlyintheprocess.Dropsofthechitosansolutionwere
extruded manually from the syringes into a Teflon 100-well plate
(homemade) containing MC at room temperature (a drop per well).