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filling and drug delivery applications, as well as facilitating that both drug-loaded TBMs significantly promoted
bone regeneration at defect regions. osteogenic differentiation in hMSCs, as evidenced by the

The elastic modulus of the materials was also measured. increased expression of osteogenic marker genes (ALP
We observed that the core markedly enhanced the and RUNX2) (Figure 2D and E). In addition, RNA-seq
crush resistance of the HAMA hydrogel, whereas the was performed on ADSCs treated with drug-loaded TBM
TBM exhibited superior elasticity and material strength (Figure 2F). After log transformation, DEGs induced by
compared to the other groups, addressing the mechanical bergamottin or the recombinant miR-138-5p antagonist
load-bearing requirements for a potential bone-filling were identified (Figure 2G). GO and KEGG pathway
material (Figure 1G). enrichment analyzes revealed that these DEGs are mainly
involved in extracellular matrix organization, ossification,
The TBM also demonstrated the ability to carry and and biomineralization processes (Figure 2H and I).
slow-release drugs. To evaluate small-molecule slow- Based on this drug-carrying capacity, we also investigated
release properties, we used rhodamine B and demonstrated
that the TBM slow-released this drug within 4 days the long-term slow-release effects of the TBM for osteogenic
(Figure 1H). In addition, the sustained release of nucleic drugs. Drug-loaded TBM was immersed in high-glucose
acid drugs was investigated using FAM-NC. Importantly, DMEM to monitor its release effects. The medium was
collected on days 7, 14, 21, and 28 to treat hMSCs. We
the TBM effectively loaded and slow-released nucleic acid observed that the TBM loaded with the miR-138-5p
drugs within 4 days. When combined with the nucleic acid antagonist consistently inhibited miR-138-5p expression in
delivery vector PVAm, the TBM’s slow-release capability
21
was markedly enhanced, with high fluorescence signals hMSCs for up to 28 d (Figure 3A). Subsequent RT-PCR,
persisting until day 14 (Figure 1I). These TBM’s slow- ALP, and ARS staining results demonstrated that the TBM
loaded with bergamottin or the miR-138-5p antagonist
release properties findings highlight its potential as a bone- promoted osteogenic differentiation over a long period.
filling material for the repair of bone defects.
Specifically, the TBM loaded with bergamottin maintained
3.2. Effect of the TBM on in vitro osteogenic osteogenic promotional effects for 21 d, while the TBM
differentiation loaded with the miR-138-5p antagonist maintained these
effects for 28 d (Figure 3B-E). These findings indicate that
We evaluated the in vitro cytocompatibility of the TBM by the TBM can slowly release drugs and exert long-term
immersing it in cell culture media and assessing its effects regulatory effects on osteogenic differentiation.
on cell activity, the cell cycle, and cell adhesion (Figure 2A).
CCK-8 results showed that the activity of hMSCs remained 3.3. Cell-embedded TBM as a potential organoid
largely unchanged over 72 h in the presence of the TBM We further investigated TBM’s potential as an organoid by
compared to the untreated group (Figure 2B). Cell cycle embedding cells into its matrix. ADSCs were used to minimize
analysis further confirmed that the TBM did not alter the the risk of immunological rejection. ADSCs were embedded
distribution of cell cycle phase in hMSCs compared to into both the TBM and HAMA hydrogel and cultured in
untreated cells or HAMA-treated cells (Figure S2A). In a flowing cultural medium (Figure 4A). CCK-8 assays
addition, hemolysis assays demonstrated that the TBM demonstrated that ADSCs embedded in the TBM remained
caused no hemolysis (Figure S2B and S2C). Collectively, highly active after 72 h (Figure 4B). EdU staining revealed
these results indicate that the TBM exhibits no cytotoxic that embedded ADSCs maintained a high proliferation rate
effects. of over 50% for 2 weeks, whereas calcein AM/PI double
To assess the TBM’s potential as an osteogenic drug staining confirmed cell viability remained at approximately
carrier, we selected the small-molecule drug bergamottin 85% during the same period (Figure 4C and D). When
and the nucleic acid drug recombinant miR-138-5p ADSCs were cultured in an osteogenic medium for 72 h,
antagonist, both of which have been previously shown alizarin red-alcian blue staining and safranin O staining
to significantly promote osteogenic differentiation. 32,33 showed that cells were distributed both within the hydrogel
Blank TBM with DMSO was used as the control group for layers and on the Porous (Figure 4E and F) of the TBM,
bergamottin, whereas MSA served as the control for the suggesting that the TBM supports osteogenic cell growth
recombinant miR-138-5p antagonist. The drugs were loaded in skeletal structures to facilitate potential trabecular bone
into the TBM, and their effects on osteogenic differentiation development. Micro-CT imaging revealed the formation of
levels in hMSCs were evaluated. ALP and ARS staining a trabecular-like structure in the ADSCs-embedded TBM
demonstrated that the TBM loaded with either bergamottin after 1 week of culture. The trabecular density increased
or the miR-138-5p antagonist markedly increased ALP markedly over 3 weeks of culture (Figure 4G), implying that
activity and mineralization nodule formation rates in ADSCs could form trabecular-like bone within the TBM.
hMSCs (Figure 2C). RT-PCR analysis further confirmed We also embedded ADSCs along with osteogenic drugs

Volume 1 Issue 2 (2025) 7 doi: 10.36922/OR025040003