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International Journal of Bioprinting Dual tuning of 3D-printed SilMA hydrogel

which further suggests that as the number of compressions due to the dispersed cross-linking network caused by the
increased, the structures, such as the polymer chains presence of PEO emulsion.
and cross-linking networks inside the hydrogel scaffolds,
gradually became stable with reduced energy dissipation. 3.4. In vitro study of cytocompatibility and cartilage
It indicates that the material had a certain degree of self- tissue engineering
adaptability and stability. When subjected to repeated 3.4.1. Live/dead cell staining
loads, it was able to ensure that the mechanical properties Compared to conventional 2D cell culture systems, 3D
were maintained and energy consumption was reduced, culture better replicates physiological microenvironments.
making it suitable for cartilage repair scenarios that involve Scaffold materials must simultaneously support long-term
bearing dynamic loads. cell survival and actively promote cartilage regeneration. 47,48
We evaluated AC performance in hydrogels through
3.3.3. Rheological performance viability and proliferation assays. Cell-laden hydrogels
The viscoelastic properties of hydrogels were rigorously were cultured for 1 and 7 days, with viability assessed via
characterized through oscillatory rheometry, providing live/dead staining. On Day 1, all groups showed sparse
critical insights into their structural integrity under viable ACs (green fluorescence) with minimal dead
dynamic loading. Strain sweep tests (Figure 5A–F) revealed cells (red fluorescence) (Figure 6A). Prolonged culture
that the storage modulus (Gʹ) exceeded the loss modulus revealed significant cell population growth, confirming
(G˝) within the strain range of 0.01–1%, indicating solid- hydrogel biocompatibility for extended cultivation. The
like behavior. The Gʹ and G˝ curves of 1%NF/SilMA, hydrogels with pores formed by PEO demonstrated higher
PEO/1%NF/SilMA, and PEO/2%NF/SilMA remained AC density than the regular controls, indicating that
parallel in this range, suggesting enhanced structural PEO incorporation significantly enhanced proliferation,
stability due to NF incorporation. However, the 2%NF/ aligning with established literature.
SilMA group exhibited a declining trend near 1% strain,
indicating partial structural disruption. Frequency sweep 3.4.2. DNA content and glycosaminoglycan deposition
tests (Figure 5G–K) showed minimal stress changes at low The proliferation of ACs within the hydrogel scaffolds
frequencies and significant changes at high frequencies. was further quantified through DNA quantification after
The increase in Gʹ for 1%NF/SilMA, PEO/1%NF/SilMA, 7- and 14-day cultures (Figure 6B). While all groups showed
and PEO/2%NF/SilMA groups confirmed improved time-dependent increases, PEO-modified hydrogels
hardness and stability with NFs. Conversely, the decrease in consistently exhibited higher DNA content than their non-
Gʹ for the 2%NF/SilMA indicated mechanical degradation. porous counterparts at both time points. These findings
The rise in G˝ in NF-containing groups suggested weak confirm that hydrogels with larger pore size and porosity
interfacial bonding between NFs and the hydrogel matrix, produced by PEO create superior 3D microenvironments
22
as well as suboptimal cross-linking, consistent with for AC proliferation. GAG deposition serves as a key
49
mechanical performance results. 20 indicator of chondrocyte functionality. Therefore,
GAG secretion of AC in hydrogels was further measured
The UV-curable hydrogel underwent a rapid formation (Figure 6C). Given that initial cell seeding density influences
of its structural network within seconds under UV light, GAG quantification, we normalized GAG values to DNA
which was effectively monitored and characterized using content to eliminate inter-sample variability (Figure 6D).
rheological methods. A transition from G’’ > G’ to G’ > G’’ On Day 7, it can be seen that incorporating PEO enhanced
was observed, indicating a shift from viscosity-dominated cell proliferation and GAG production but adding NFs
to elasticity-dominated behavior in the hydrogel had no noticeable effect. This trend, induced by PEO
(Figures 5K and S6). The gelation times for different groups modification, was intensified with prolonged culture on
were summarized based on the crossover points in the local Day 14. At that time, the introduction of NFs also exhibited
plots (Figure 5L). Notably, the gelation times of 1%NF/ a significant positive effect on GAG deposition, especially
SilMA and 2%NF/SilMA groups showed no significant for the 1% concentration. The enhanced performance of
difference compared to the SilMA group, which suggested porous hydrogels might be attributed to PEO-induced
that the 30% SilMA hydrogel inherently possesses a porosity, which facilitates oxygen and nutrient transport.
50
densely crosslinked network, and the addition of NFs does Additionally, the exceptional GAG deposition observed in
not accelerate the gelation process. Jin et al. have reported the PEO/1%NF/SilMA group could be attributed to the
46
that excessive incorporation of nanomaterials may disrupt homogeneous distribution of NFs and larger pores within
the curing system of hydrogels. 33,46 In the case of PEO/ the hydrogel matrix, which modulated ACs’ behavior at the
SilMA hydrogels, the extended gelation time is likely microscale.

Volume 11 Issue 4 (2025) 289 doi: 10.36922/IJB025140118