Our cytotoxicity findings are consistent with several other studies
using GO layer cultured with mesenchymal stromal cells and GO/chitosan
scaffolds seeded with human adipose-derived stem cells [53, 54]. In
our study, LDH measurements showed significantly higher levels of DPSCs
toxicity for uncoated SA scaffolds which can be attributed to poor
ability and lack of efficient sites to support cell adhesion and
proliferation. This result is supported by another study utilizing other
materials mixed with alginate to create biocomposites [55]. However,
the incorporation of GO into GOSA scaffolds did not elicit significant
differences in cytotoxic effects after 2 days of DPSCs culture. Overall,
the GO-enriched scaffolds exhibited cytotoxicity of 15–27% after 48h
of culture, suggesting that these materials are biocompatible with
DPSCs. Therefore, GO has no apparent cytotoxic effect but
exhibits positive effects on cell
function in long-term DPSCs culture.
The use of serum-free or serum-rich culture media for biological assays
is a contentious issue [56]. The associated clinical uncertainties
with the use of FBS include immune rejection, batch-to-batch variations,
ethical concerns, interfering effects of unidentified growth factors and
proteins, and viral contamination [57]. Regarding DPSCs stability,
there is clear evidence about the positive effects of serum-free culture
media on retaining stemness [58, 59]. Furthermore, serum-free
cultured DPSCs within chitosan scaffolds expressed stem cell markers
(Nestin and Sox2) and survived successfully after transplantation into
the rodent spinal cord [60]. The present study investigated whether
serum depletion facilitates the attachment of DPSCs onto hybrid
scaffolds. Thus, the effects of serum on the cytotoxicity of DPSCs were
tested using 2D and 3D culture systems across three different coating
conditions at a single cell density of 4 × 104cells/sample. We have indicated that the presence of serum caused
significantly higher cytotoxic behaviour of coated 3D scaffolds which
could be due to suboptimal surface-cell attachment [20].
This was also shown for the
combination of PLL and LAM coatings in the presence of serum on 3D
structures. It is surmised that coatings lower the available surface
area of 3D scaffold biomaterials for the attachment of cell receptors,
which subsequently causes poor adhesion. In contrast, coated 2D surface
was found to result in better attachment of DPSCs which is due to the
unfavourable bare tissue culture surface. Moreover, our study found that
the cytotoxicity of DPSCs cultured in serum-rich media on 3D scaffolds
increased significantly in comparison with the 2D control. The increased
level of cytotoxicity in serum-containing medium could be attributed to
the surface oxygen content of GO and RGO, which favours FBS adsorption.
This could be due to the formation of protein corona on the GO surface
which in turn influence the toxicity of GO-based materials [61, 62].
Furthermore, studies have indicated that serum proteins can have an
interfering effect on interactions between nanoparticles, cells and
biological molecules [63-65]. Accordingly, Lesniak et al. [64]
demonstrated that various protein corona formed on silica nanoparticles
modified cell adhesion, cellular uptake, and toxicity, which is
determined by the serum protein concentration. It was also shown that
serum-containing media resulted in lower cell adhesion and
internalization efficiency of silica nanoparticles. The present study
found DPSCs can be seeded in serum-free media onto GOSA and RGOSA
scaffolds with no cytotoxic effects, showing promising potential for
clinical translation as cellular transplants are typically serum-free.