Discussion
Significant complexity has been
uncovered in the interaction between stem cells and engineered scaffolds
[38]. In this study, we report the profiles of DPSCs cultured on
fabricated 3D GOSA and RGOSA scaffolds with 2D, coating and media
controls.
Our in vitro biodegradation data revealed an inverse relationship
between GO concentrations and weight loss. This relationship is
explained by accessibility of water molecules to GO composites as a
function of GO concentration. As discussed in the water contact angle
measurements in our previous paper [24], the incorporation of GO
increases the interaction of
composite
scaffolds with water and this effect is GO concentration-dependent.
Therefore, as hydrophilicity accelerates with higher GO concentration,
water-mediated scaffold degradation increases [39]. Notably, a
reduced degradation rate is assumed to be beneficial for tissue
regeneration [40]. Collectively, our results confirm that the
degradability of composite 3D R/GOSA scaffolds is adjustable and
controlled by graphene content.
It is noteworthy that when cultured onto 3D SA and GOSA scaffolds, DPSCs
viability was enhanced in relation to 2D culture plates. This
relationship clearly signifies the optimal condition of initial cell
adhesion to the scaffold surface to promote subsequent cell
proliferation and infiltration. Our observation of an increase in the
total metabolic activity of cell-seeded scaffolds provides
proof-of-principle support for cell growth and proliferation in a 3D
matrix which can act as a delivery system for seeded cells. Thus, it is
reasonable to conclude that an artificial 3D scaffold is an acceptable
approach to mimic the natural architecture of the native tissue and
crease a microenvironment conducive to DPSCs engraftment.
The cell-cell interaction within the matrix of 3D cell culture systems
has a profound influence on cellular functions including viability,
migration and proliferation in contrast to 2D culture [41]. For
example, one report showed that 3D polymer-based scaffolds seeded with
hepatic cells had less cytotoxic effects than those cultured in 2D
[42]. In another study, the 3D culture of dental stem cells was
found to support their neuronal characteristics and maintain cell
phenotypes [43]. Extending this, we have confirmed that a superior
proliferative ability of DPSCs, as measured by metabolic activity, can
be obtained when cells are cultured on 3D porous scaffolds.
Regarding cell seeding density, we have shown that when cells are seeded
on 3D scaffolds at all densities, the cell proliferation rate is
significantly increased in comparison to 2D. Moreover, we found that
increased seeding density in 3D scaffolds could be achieved without
inducing cytotoxic effects, as determined by LDH assay. In this study,
we also showed that the addition of graphene to 3D composite scaffolds
improved cellular behaviours and this was seen across all DPSCs seeding
densities examined. Notably, the degree of cellular metabolic activity
did not differ significantly between the different cell densities
tested. This result is important because the implication is that seeding
efficiency can be achieved even at high cell densities, at least within
a 48-hour period. More work would be required to determine if high
seeding densities might have different effects on longer-term culture.
Assessment of coating reagents revealed that PLL+LAM coating did not
affect cell viability as indicated by AB reduction percentage. After 48
hours of DPSCs culture, both laminin coating and no coating conditions
decreased cell viability on both SA and GOSA scaffolds. Possible
explanations involve interactions between coating properties and DPSCs
adherence or cell aggregation which can decrease proliferation [44].
Interestingly, PLL was identified as the coating reagent that enhanced
cell-matrix adherence. This
enhancement might be due to the larger number of cationic sites offered
by PLL coating on the 3D surface. In agreement with another study
[44], our results show that PLL is superior to laminin coating. In
addition, the effect of all three coating conditions on DPSCs was
irrespective of cell seeding density.
The AB assessment of metabolic activity shows that biomaterial
composition can modulate DPSCs responses to fabricated scaffolds. Our no
scaffold controlled LDH results also indicate that SA, GOSA and RGOSA
scaffolds materials are nontoxic to DPSCs in short-term culture. Based
on previous work, it might be inferred that the composition of a
scaffold material can have a direct effect on the biodistribution of
secreted factors that in turn influence the stem cell fate. The results
appear to suggest that different scaffolds with varying material
properties (such as blend ratio, swelling index, or microstructure)
elicit diverse DPSCs behaviours. The increase in cell viability observedin vitro upon the incorporation of graphene in composite
scaffolds is consistent with other studies [36, 45]. Published data
suggest that the outstanding surface properties and adsorption capacity
of graphene-based nanomaterials are the main contributors to the
observed DPSCs responses.
Our results showed a strong influence of pore size, material composition
and substrate dimensionality on cell viability, in accordance with the
findings of Domingos et al. [46]. Comparisons of AB reduction in SA
(97.2%) and graphene-based scaffolds including GOSA0.5 (97.5%), GOSA1
(98.0%), RGOSA0.5 (99.05%), and RGOSA1 (99.18%) revealed the
differences in DPSCs proliferation markers across various graphene-based
scaffolds. These differences appear to be explained by variations in
scaffold porosity (%) such that scaffolds with higher porosity (RGOSA ≈
99%) are able to accommodate higher numbers of viable cells.
Furthermore, it was shown that scaffolds with smaller mean pore sizes
induce relatively less toxicity. This can be explained by the available
surface area of scaffolds for cultured cells or applying the principle
that the mean pore size and specific surface area are inversely
proportional. In consideration of the specific surface area and mean
pore size of a scaffold, biophysical properties can affect cell adhesion
[47]. It follows that the low levels of cell adhesion are observed
on scaffolds with larger pore size and less specific surface area [48,
49]. As a result, the available specific surface area per unit volume
for cell adhesion of each fabricated scaffolds can be calculated using
mean pore sizes [50]. Accordingly, the normalized specific surface
area of GOSA and RGOSA scaffolds, as shown in Table 1, can be obtained
by dividing the mean pore size of each scaffold by mean pore size of SA
scaffold (3D control). Thus, the higher AB reduction observed in our
RGOSA1 scaffold can be explained by the higher specific surface area in
comparison with GOSA1 and SA scaffolds. These data indicate that higher
pore size facilitates increased DPSCs migration and proliferation, in
agreement with a previous report on culturing DPSCs into 3D
poly-L-lactic acid-based scaffolds [51]. In addition, the mechanical
properties of scaffolds with overly large pores are compromised, whereas
higher cellular proliferation within large pore sizes can have
implications for differentiation [52].
Table 1. Estimates of the specific surface area of graphene-based
scaffolds relative to SA scaffold