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Global Translational Medicine Gelatin-based cell carriers for tooth-germ organoids

GelMA HG possesses a crosslinked structure that forms metabolic rates, which may contribute to the higher
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a bulk HG, which may hinder effective nutrient and waste metabolic activity on GelMA MS. Factors such as size
exchange between embedded cells and their surrounding distribution, porosity, and surface topography likely
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environment. In contrast, cells in GelMA MS and Gel influence the rate and effectiveness of nutrient exchange
MS exhibited an elongated morphology and spanned within the constructs, as well as the available surface area
between microparticles (Figure 3A[i, iii, v, vii]), whereas for cell adhesion and growth. 32,33 These characteristics may
cells in GelMA HG and maintained a non-elongated differ between carriers due to their respective fabrication
morphology (Figure 3A[iv and viii]). This cellular methods and crosslinking agents. 34
arrangement promotes aggregation, suggesting enhanced Cell viability in GelMA HG may be underestimated due
cell-cell contact and communication compared to GelMA to limited reagent permeability in and out of the HG in the
HG, where cells were only elongated on the HG surface metabolic assay (Figure 3B). Conversely, DNA measured
and remained rounded within the bulk HG. Studies have from dissolved GelMA HG might include DNA from
shown that cell-cell communication is important for the dead cells trapped in the HG, potentially overestimating
viability and functionality of hDPSCs in tooth organoid cell numbers. Although neither assay provides a definitive
formation. Furthermore, the observed 3D structure measure of cell growth on GelMA-based carriers, both
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and cell spreading likely enhance both cell-cell and cell- consistently showed that Gel MS supported the highest
scaffold mechanical interactions, which are essential for hDPSC growth, which was consistent with phase image
maintaining stem cell viability. 30 observations (Figure 3A[v-viii]).

Cell viability was further quantified using the
alamarBlue assay to assess metabolic activity (Figure 3B). 3.3. Osteogenic differentiation of hDPSCs on/within
Notably, cells on Gel MS exhibited significantly higher different cell carriers
metabolic activity than those on GelMA MS or GelMA To support tooth regeneration, hDPSCs in organoid
MP. Between GelMA MS and GelMA MP, which share cultures undergo lineage-specific differentiation.
7,35
the same material composition, GelMA MS supported Osteogenic and odontogenic differentiation are commonly
significantly higher viability. Although GelMA MP is used to assess the differentiation potential of hDPSCs
expected to provide more surface area than spherical in tooth tissue applications, as these cell types share
GelMA MS, the lower metabolic activity observed suggests similarities, and hDPSCs have been shown to be capable of
that factors such as surface curvature may influence cell differentiating into both lineages. To assess the osteogenic
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adhesion, proliferation, or metabolism beyond surface differentiation potential of hDPSCs on/in various carriers,
area alone. Among all four carriers, GelMA HG showed cell aggregates were cultured in osteogenic medium
the lowest cell viability. for 10 days. These organoid-like aggregates, both with
Since the alamarBlue assay may not accurately reflect and without osteogenic induction, were then stained
cell counts due to reagent permeability limitations in with alizarin red solution (Figure 4). Alizarin red stains
aggregates or HG, DNA quantification was conducted calcium deposited by osteogenically differentiated cells.
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as an additional measure. After treating the samples Osteogenically-induced samples (Figure 4A, upper panel)
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with type I collagenase to dissolve aggregates or the HG, displayed significantly stronger red staining compared
released DNA was measured using the Helixyte DNA assay to uninduced samples (Figure 4A, lower panel), with the
(Figure 3C). Consistent with the metabolic activity results, exception of GelMA HG. Interestingly, GelMA HG without
Gel MS supported the highest cell growth based on DNA cells also showed strong staining (Figure 4A), indicating
content. Interestingly, DNA quantities on GelMA MS, possible non-specific dye binding or inefficient dye elution
GelMA MP, and in GelMA HG were similar. The similar from GelMA HG despite extensive washing steps.
DNA levels between GelMA MS and GelMA MP indicated In aggregates, intense red staining was localized to areas
comparable cell numbers on these two carriers. This result where cells aggregated between particles (Figure 4A[i–iii]),
may highlight the differential effects that size, crosslinking while in GelMA HG, staining was more pronounced on the
methods, and geometries have in promoting cell viability. surface where elongated cells were located (Figure 4A[iv]).
Although the reason for the higher metabolic activity of This pattern aligns with viability assays, which indicated
cells in GelMA MS compared to GelMA MP (Figure 3B) higher viability of cells grown on GelMS MS, GelMS MP,
remains unclear, Calcein AM staining revealed elongated and Gel MS throughout organoid-like structures, compared
cells on the surfaces of GelMA MS (Figure 3A[i]), whereas to the lower viability of hDPSCs encapsulated in GelMA
staining on GelMA MP appeared punctuated and discrete HG. These findings suggest that cell-cell interactions and
(Figure 3A[ii]). Increased cell spreading is known to raise elongation facilitated differentiation, as these phenomena

Volume 4 Issue 1 (2025) 75 doi: 10.36922/gtm.5897