3.1 Agarose-collagen hydrogel: preparation and characterization
Three agarose-collagen (A-C) weight ratios were used for preparation of
the hydrogels used in this paper, i.e. 0.5%-0.02%, 0.25%-0.02%, and
0.125%-0.02%, respectively. The collagen amount (equal to 0.2 mg/mL)
was kept constant in all the hydrogel formulations. This concentration,
close to that used in other studies in which blended hydrogels were
prepared, is sufficient to provide the anchoring sites to the embedded
cells. On the other hand, the amount of agarose was varied from 0.5 to
0.125% to evaluate the impact on the biophysical properties of the
hydrogel and consequently their effect on the cellular spheroids
development. To prepare the hydrogels and, at the same time, to prevent
the denaturation of collagen and the cell damage and death, the agarose
solution was first heated until boiling (100 °C) to dissolve the
polysaccharide, and then cooled down until it reached 45 °C. At this
point, since gelation of pure agarose occurs at around 36 °C, it was
rapidly mixed with collagen and the cell suspension. The preparation
sketch is reported in Figure 1S.
The physical properties of the A-C hydrogels were characterized. The
study of the hydrogels surface structure and topology through SEM
techniques shows that decreasing the agarose percentage, the porosity of
the hydrogels increases and the whole structure appears less compact
(Figure 1). In 0.125%-0.02% A-C hydrogels, indeed, the higher
percentage of water makes the dry structure very brittle displaying
pores that are quite uniform and interconnected with a mean size of 71 ±
14 µm (Figure 1e and f). Although the measure of the pore sizes of a dry
structure cannot be considered realistic, it provides an estimation of
the 3D organization of the hydrogels. In 0.25%-0.02% and 0.5%-0.02%
A-C hydrogels, the number of pores is smaller, while the average pore
size appears to be slightly larger (81 ± 21 and 87 ± 25 µm,
respectively, Figure 1a-d). The higher turgidity and compactness of the
hydrogels with higher percentage of agarose is also evident at a
macroscopic level, as shown in the bottom panel of Figure 1S. Indeed,
while 0.25%-0.02% and 0.5%-0.02% A-C hydrogels appear self-standing,
that with 0.125%-0.02% weight ratio looks softer and less compact.
FTIR spectroscopy was performed on the hydrogels to investigate whether
collagen, though the low percentage amounts used, was detectable on the
outer surface of the hydrogels, thus confirming its distribution within
the hydrogel texture. To this aim, the analysis was performed directly
on the synthetized hydrogels deposited on the ATR crystal, without any
further manipulation. Pure agarose hydrogel (0.25%) was recorded as
reference (light green line in Figure 2a), showing the typical signals
at 988 and 1076 cm-1, relative to the C-H bending and
to the C-O stretching of the glycosidic bonds, and two broad peaks at
1656 and 3421 cm-1, characteristic of the stretching
of the H-O-H bound water and of the O-H hydrogen bonded carbohydrate
hydroxylic groups, respectively (Oza,
Prasad, & Siddhanta, 2012). Two broad peaks were also observed in the
pure collagen hydrogel at about 3328 and 1645 cm-1 for
the amide C=O and N-H stretching, respectively (pink line in Figure 2a).
Additional smaller signals were detected at 1051 cm-1for the C-OH stretching vibrations of carbohydrate moieties attached to
the protein (Petibois, Gouspillou, Wehbe,
Delage, & Déléris, 2006). The resulting FTIR spectrum of the blend
0.25%-0.02% A-C hydrogel (red curve of Figure 2a) showed all the peaks
characteristic of the pure compounds, i.e. smaller signals at 989 and
1079 cm-1, with a small side-bump at 1045
cm-1, and much broader peaks at 1649 and 3464
cm-1. Interestingly, while the last two peaks had
similar intensity in pure collagen, a much higher intensity of the
signal at lower frequencies was observed in pure agarose as well as in
the blend hydrogel. As expected, no significant differences were
observed in the spectra of the other two blend hydrogels (data not
shown).
Another critical feature of a hydrogel is the capability to absorb and
retain water. Figure 2b) shows the swelling behavior of the three types
of hydrogels. The hydrogels containing 0.5% and 0.25% agarose show a
similar trend with a swelling ratio respectively of 20 and 25 times at
t0 and a maximum swelling of 27 and 30 times after 24 h
in PBS. These data are in accordance with those reported in the
literature. On the other hand, the ability to absorb water of the
hydrogel with 0.125% agarose is considerably lower, with a swelling
ratio of 5.2 times at t0 and 7.1 at t24,
respectively. Thus, there appears to be a critical threshold of agarose
percentage below which the physical properties of the hydrogel are
dramatically altered.
A degradation test was carried out to investigate the stability of the
three formulations over time. Panels c) and d) of Figure 2 show the
percentage residual weight of the hydrogels kept at 37 °C in PBS and
DMEM up to two weeks. 0.5%-0.02% and 0.25%-0.02% A-C hydrogels were
shown to be quite stable to degradation and lost around 10 and 15% of
their weight after 14 days of incubation in PBS and DMEM with 10% FBS,
respectively. On the other hand, 0.125%-0.02% A-C hydrogels showed a
maximum of degradation close to 40% after 2 weeks in both incubation
media.
In parallel, the amount of collagen potentially released was estimated
via a protein quantification assay. To this aim, the A-C hydrogels were
kept in PBS at 37 °C and the volume of PBS was collected and renewed
every 24 h up to 14 days. Figure 2S shows the results of the BCA assay
carried out with the three types of hydrogel. Collagen release was
detected in all hydrogels but to a higher extent and with a quicker
trend in that with a lower agarose content. The overall percentage
amount of collagen release after 2 weeks is equal to 50%, 27%, and 8%
of the whole collagen present in the hydrogel containing 0.125%,
0.25%, and 0.5% agarose, respectively. This loss, together with the
less compact texture of the 0.125%-0.02% A-C hydrogel, would explain
the higher degradation of this matrix.
A further parameter that deserves investigation is the capability of a
hydrogel to allow the diffusion throughout the matrix of biomolecules
and nutrients, such as growth factors and serum proteins having
molecular weight of several tens of kDa. To this aim, a diffusion test
was performed by using two fluorescent probes, FITC-conjugated
hyaluronic acid and Transferrin-TRITC, with a MW of around 10 and 80
kDa, respectively. Once loaded onto the three hydrogel formulations, the
variation of concentration of the feeding solution was monitored over
time and reported versus the diffusion in pure PBS. The results show
that hyaluronic acid (Figure 2e) diffuses rapidly into the softest
hydrogel reaching 100% diffusion after 72 h, while the other two
hydrogels show a similar trend although with a more gradual diffusion
that decreases by increasing the percentage of agarose, reaching a
maximum of 82% and 75% after one week, respectively. On the other
hand, in the case of transferrin-TRITC the diffusion is much slower: in
the 0.125%-0.02% A-C hydrogel the diffusion reaches almost 48% after
24 h, while at the same time point it is closed to 10% in the case of
the other two blends. (Figure 2f) Noteworthy, after one week the
diffusivity does not exceed 50% even in the softest matrix.
The structural properties of the A-C samples were also evaluated by mean
of compression test under physiological-like conditions (PBS, 37 °C).
Figure 3a) shows how the agarose concentration deeply affects the
hydrogels’ mechanical properties. In fact, the increase in agarose
concentration corresponds to an increase in the compressibility modulus.
As expected, the 0.5%-0.02% ratio shows the highest E modulus (5.7 ±
0.5 kPa), followed by 0.25%-0.02% (1.6 ± 0.4 kPa) (p = 0.0004) and
0.125%-0.02% (0.7 ± 0.2 kPa) (p = 0.0001). A minor but significant
difference was found between 0.25%-0.02% and 0.125%-0.02% (p =
0.03). In order to correlate degradation resistance to structural
stability over time, the A-C samples’ mechanical performances were
evaluated after 0, 1, 4, and 8 days of incubation in physiological-like
conditions (PBS, 37 °C, in humified atmosphere with 5%
CO2). While 0.125%-0.02% hydrogels could not be tested
over time due to their low consistence, the 0.5%-0.02% and
0.25%-0.02% blends retained their structural integrity until the
8th day of measure. As shown in Figure 3b, no
significant changes in the E modulus were registered in the case of
0.5%-0.02% and 0.25%-0.02% hydrogels. These data are in accordance
with the degradation tests in which a minimum weight loss was recorded.