Tripolyphosphate

Chitosan
scaffolds were prepared as described with slight modifications to
the protocol.^{24 (link)} Briefly, 1% chitosan gel
was prepared by dissolving chitosan in deionized water containing
1% acetic acid under a continuous stirring condition for 48 h. The
gel was centrifuged at 3350 RCF to pellet-undissolved chitosan flakes,
and a clear supernatant was used for scaffold fabrication. The gel
was aliquoted by weight (750 or 2000 mg) in 5 or 10 mL scintillation
vials, respectively. Rapamycin (1 or 50 μg) or tetracycline
(10 or 100 μg) was added to make drug-loaded scaffolds. The
samples were vortexed thoroughly and frozen at −80 °C
for a minimum of 3 h. The samples were lyophilized for 48 h using
a freeze-dryer (TAITEC, Japan). Sterile crosslinked scaffolds were
prepared by treating the lyophilized gels with 1 mL of sterile 5%
tripolyphosphate (Merck) at pH 5 for 5 min followed by two washes
using sterile 1× PBS.
To determine the swelling characteristics,
tripolyphosphate crosslinked scaffolds were weighed and immersed in
2 mL of 1× PBS and kept at 37 °C. At various times, the
weight of the scaffold was measured after approximately 5 min of drying
to remove excess water. Following weighing, the scaffolds were immersed
in freshly replenished 1× PBS. The change in weight at every
time point was determined by normalizing the value to the original
weight of the scaffold.