Abstract:
The investigation of natural bioactive injectable composites to induce angiogenesis during
bone regeneration has been a part of recent minimally invasive regenerative medicine strategies.
Our previous study involved the development of in situ-forming injectable composite hydrogels
(Chitosan/Hydroxyapatite/Heparin) for bone regeneration. These hydrogels offered facile rheology,
injectability, and gelation at 37 ◦C, as well as promising pro-angiogenic abilities. In the current study,
these hydrogels were modified using glycerol as an additive and a pre-sterile production strategy
to enhance their mechanical strength. These modifications allowed a further pH increment during
neutralisation with maintained solution homogeneity. The synergetic effect of the pH increment
and further hydrogen bonding due to the added glycerol improved the strength of the hydrogels
substantially. SEM analyses showed highly cross-linked hydrogels (from high-pH solutions) with a
hierarchical interlocking pore morphology. Hydrogel solutions showed more elastic flow properties
and incipient gelation times decreased to just 2 to 3 min at 37 ◦C. Toluidine blue assay and SEM
analyses showed that heparin formed a coating at the top layer of the hydrogels which contributed
anionic bioactive surface features. The chick chorioallantoic membrane (CAM) assay confirmed significant enhancement of angiogenesis with chitosan-matrixed hydrogels comprising hydroxyapatite
and small quantities of heparin (33 µg/mL) compared to basic chitosan hydrogels.
