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International Journal of Bioprinting Fine collagen scaffold for osteogenesis

hydrogel scaffolds used in this study were viscoelastic and have contributed to the upregulated expression of NCAD
hydrophilic, and therefore, more favorable for cell adhesion and HIF-1α. In addition, the expression of β-catenin was
and migration. 63–65 Additionally, the collagen I composition also highest in sample 2, and the upregulation of this factor
had the surface RGD groups that guided and promoted cell has been shown to promote osteogenic differentiation in
adhesion, and it was also superior in nutrient adsorption multiple studies. We believe these findings could deepen
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during cell culture. Presumably due to these reasons, the our understanding of how scaffold dimensions and
use of the collagen I-based hydrogel could “switch” the structures influence osteogenic differentiation and shed
optimum pore size to a smaller dimension. light on the possible mechanisms involved.
In recent years, gelatin has been routinely used as a 4.4. Broader impact of our study in bone tissue
key component for producing hydrogel scaffolds, as it engineering
is a naturally derived protein with a surface RGD group We believe the findings of this study offer several important
like collagen. 14,26,29,66–70 Though gelatin-based scaffolds contributions to the field of bone tissue engineering. First,
have shown significantly improved outcomes in cell our results provide insight into resolving the long-standing
adhesion and proliferation compared to metal and trade-off between cell proliferation and osteogenic
thermoplastic biopolymers, 14,71 they remain inferior to differentiation in scaffold design. 62,68,74,80 Conventionally,
scaffolds containing collagen I for bone tissue engineering. smaller pores (100–150 μm) are known to promote
Compared to gelatin, collagen I is a native component of osteogenic differentiation, but they often hinder cell
bone and has been shown to have a greater osteogenic- proliferation. In bone tissue engineering, both factors
promoting effect. 5,72,73 Therefore, we achieved a high are important: while higher proliferation increases the
printing resolution with a hydrogel ink containing 8% w/v total production of ECM components, differentiation
collagen I, which improved the biochemical and structural determines the proportion of bone-specific ECM
properties of the scaffolds and could synergistically components. 81,82 More importantly, our results indicated
promote osteogenic differentiation of MSCs. Therefore, that higher cell proliferation may actually promote
our work steps further than recent studies utilizing gelatin- osteogenic differentiation by upregulating the expression
based scaffolds. of NCAD and HIF-1α. This highlights the importance

4.3. Possible mechanism related to the effect of of addressing both proliferation and differentiation in
scaffold structure on osteogenic differentiation tandem rather than in isolation. In this study, such a trade-
To our best knowledge, studies on the effects of structure off was successfully solved in sample 2, which exhibited
on osteogenic differentiation are generally lacking in mesh the highest levels of both proliferation and osteogenic
scaffolds. In the few studies applying scaffolds with relatively differentiation, indicating that an optimal pore size can
support both processes simultaneously.
big pore sizes (300–700 μm), the level of osteogenic
differentiation increased monotonically in smaller pores Second, our work addresses a key challenge in
within this range. However, it is still necessary to produce scaffold development: the difficulty of optimizing both
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scaffolds with much higher resolution to further explore the compositional and structural properties for bone
their potential to promote osteogenic differentiation and regeneration. Although collagen I is widely regarded as
investigate the possible underlying mechanisms. one of the most favorable biomaterials for bone tissue
83
In this study, the level of osteogenic differentiation engineering, previous studies have either failed to
was higher in sample 3 than in sample 4, indicating the fabricate high-resolution collagen I-based mesh scaffolds
advantage of using collagen I over gelatin. Among the or have achieved high-resolution scaffolds using alternative
6,84,85
collagen I-based scaffolds, sample 2 led to the highest level materials such as bioplastics, metals, or ceramics.
of osteogenic differentiation compared to samples 3 and 4. In our study, we addressed the printability limitations of
collagen I-based hydrogels by introducing a Schiff-base
According to our WB test results, the expression of NCAD interaction, which enhanced ink rheology and enabled
was the highest in sample 2, indicating the highest level of cell the successful printing of high-resolution scaffolds. This
condensation and cell–cell communication, which could allowed us to simultaneously optimize both composition
promote osteogenic differentiation, as reported in existing and structure, enhancing the scaffold’s performance in
studies. 75–77 Additionally, the expression of NCAD was also supporting bone regeneration.
the highest in sample 2 and maybe another contributor
to promoting osteogenic differentiation. Since MSC Lastly, much of the recent progress in bone tissue
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proliferation was highest in sample 2, it presumably led to a engineering has focused on biochemical enhancements,
higher cell density, which promoted cell condensation and such as incorporating novel growth factors to stimulate
the formation of a hypoxic condition. These factors may osteogenesis. 86–89 In contrast, studies focusing on structural

Volume 11 Issue 4 (2025) 236 doi: 10.36922/IJB025140116