4.1 Glyoxal as chemical cross-linker in a hydrogel system
A straightforward method for chitosan-based solution to form permanent hydrogel
networks is chemical cross-linking. Cross-linked chitosan networks can be prepared
using the available –NH2 and –OH chemical handles and cross-linkers that can form a
number of linkage chemistries such as aminecarboxylic acid bonding and Schiff base
formation [11, 68, 69]. In general, the networks of chitosan-based hydrogels can be
formed by using small molecule cross-linkers or enzymatically cross-linkers, polymer–
polymer reactions between activated functional groups and photosensitive agents [70].
For small molecular cross-linkers, including glutaraldehyde, formaldehyde,
diisocyanate, ethyleneglycol diglycidylether (EGDE), and others, it has been many
years using these chemical cross-linkers to reinforce the mechanical strength of
hydrogels [11, 68, 69, 71]. Though these hydrogels can offer desirable properties, the
main drawbacks of small molecule cross-linkers are potential toxicity and residual
un-reacted small molecular cross-linkers [72]. Previous study reported that Genipin, a
naturally derived chemical compound from the gardenia, showed great biocompatibility
and low cytotoxicity [73]. Nevertheless, the gelation time is at least one hour [74]which
is not suitable for clinical use and an anti-angiogenesis effect of genipin may decrease
the rate of wound healing [75]. Glutaraldehyde can crosslinked chitosan chains to form
hydrogels within one hour ; however, it is considered toxic for respiratory tract, eyes
and skin. For the convenience of clinical use, we use glyoxal as chemical cross-linker in
chitosan-based hydrogels so that the networks of hydrogels can form in short time.
Recently, glyoxal has been used as an alternative dialdehyde cross-linker in various
biomedical studies and applications and is considered safe [76, 77]. Moreover, previous
study revealed that glyoxal has been shown to be cytocompatible and support viability
of the cells [78]. This chemical cross-linker can be expected to be applied in tissue
engineering and protein/drug delivery.
4.2 Rheological studies
Compared with CS-GE hydrogels crosslinked with 0% glyoxal and 0.0025% glyoxal,
CS-GE hydrogels crosslinked with 0.005% glyoxal and 0.01% glyoxal revealed a higher
elasticity degree in their rheological behavior, and they could be classified as strong
hydrogels [79-81]. The strength of hydrogels mainly resulted from contemporary
presence of physical interactions between chitosan chains, gelatin chains and β-GP,
secondary bonds such as Van der Waals forces among polymers, and chemical
crosslinks between chitosan/gelatin chains and glyoxal [82]. Obviously, glyoxal
influenced the mechanical properties of the hydrogel by increasing the shear storage
4.3 HS68 cells migration
Previous research had found that HPL contained abundant of growth factors, such as
EGF, TGF-β1, PDGF-AB, PDGF-BB, and so on. Moreover, PDGF-BB had been
proved to participated in the migration of HS68 cells [83]. In present study we
examined the wound healing effect of HPL and it revealed that HPL could promote the
migration of HS68 cells in a dose-dependent manner. Based on previous researches, we
thought the reason may be abundant growth factor, PDGF-BB, in HPL.
4.4 HUVEC migration
As our result showed on Fig. 11, our study indicated that HPL have the power to
promote the migration of HUVEC. A previous study revealed that PDGF and vascular
endothelial growth factor (VEGF) are pivotal to the formation of capillary structures
[84]. While VEGF mainly regulates endothelial cells, PDGF signaling is crucial for
cells of the vascular wall, i.e., pericytes and smooth muscle cells [85]. In addition, HPL
comprises about PDGF-AB (about 1.5ng/mL), PDGF-BB (about 3.5ng/mL), and other
growth factors [53]. Due to the induction of PDGF-AB and PDGF-BB, the migration of
HUVEC are facilitated in our study.
4.5 HUVEC tube formation
Through the activation of compound that can stimulate angiogenesis, HUVEC may
differentiate into capillary-like structure [86]. The differentiation process involves
several steps in blood vessel formation, including cell adhesion, migration, alignment,
protease secretion, and tube-like structure formation [87]. Our result showed that tube
do not form after seeding HUVEC on ECM gel 6 hours and 24 hours under condition of
2% HPL or lower since the activation of compound that can stimulate angiogenesis is
not enough. According to previous study, it was reported that epidermal growth factor
(EGF) could stimulate the tube formation of HUVEC in a concentration-dependent
manner [88]. In our result, the 5% HPL group showed denser and stronger
tube-like structures no matter on 6 hours or 24 hours and we proposed that the reason
may be sufficient promotion effect exerted by EGF.
4.6 3D human skin equivalent model
Previous study indicated that Keratinocyte secrete angiogenic growth factors, such as
VEGF and PDGF[89]. Therefore, we seeded HaCaT cells on the second layer.
According to our result, it revealed that HUVEC already formed tube-like structures
before we add the hydrogels and HPL into the wound in this model. To solve the
problems we encountered, there are some modifications need to do. First, the
composition of medium in seeding HUVEC should be modified to slow down the rate
of tube formation. Second, we need to cut the time between seeding HaCaT cells and
4.7 CAM assay
Though we found that hydrogels combined with HPL could induce the abundant
formation of vascular network in some cases, there are still some limitations. Since we
opened a window on the side of eggs , the view we observed are limited. Moreover, the
disturbance from embryo was possible to result in the move of oring and hydrogels. The
view we observed was easy to be interfered with egg shells so that it’s hard for us to
capture the oring without egg shells. Since the eggs without treatment were able to form
the vascular network themselves, it was not an easy way for us to observe prominent
difference between the control and experimental group. Some modifications of this
assay can be expected in the future. We can try to inject lens culinaris agglutinin (LCA)
and use the microscope to visualize the microvasculature.