Page 512 - IJB-10-4

International Journal of Bioprinting 3D-printed variable stiffness scaffolds

structure was investigated by pre-treating scaffolds to facilitate cell-on-cell interaction and cartilage formation.
to −20°C, −80°C, or flash-freezing in liquid nitrogen. From these findings, it is evident that pre-freezing to
Scaffolds were fabricated using a freeze-drying protocol −20 C for 2.5 h before freeze-drying resulted in pores that
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adapted from Murphy et al., whereby an annealing step are suitable for meniscus tissue regeneration, displaying
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was introduced to the freeze-drying cycle for producing improved interconnectivity across groups and attaining
scaffolds with larger pores. pore sizes of 81–163 µm.
The pore sizes varied considerably in this study with 3.7. In vitro assessment of the ECM-infiltrated
changes in the pre-freeze temperature. From the SEM PCL scaffold
images (Figure 5B and C), the pore size for GelMA was For successful meniscus cartilage tissue engineering,
found to significantly decrease (from 160 to 99 µm) when it is critical to control the phenotype of hMSCs and the
the pre-freeze temperature was altered from −20 to −80°C. composition and organization of the ECM they produce. To
A similar trend was observed with the addition of CS/ assess the chondro-inductivity of the scaffolds, hMSCs were
HAMA, whereby the pore size decreased from 163 to 98 µm statically cultured in chondrogenic media in low oxygen
as the pre-freeze temperature changed from −20 to −80°C. conditions (5% O ), which was previously demonstrated
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Incorporating CS/HA into a GelMA hydrogel caused a to support cartilage formation. The viability of hMSCs
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significant decrease in pore size compared to GelMA and on the hybrid meniscal scaffolds at D1 and D21 was
GelMA/CS/HAMA at −20°C. Flash-freezing hydrogels evaluated using Live/Dead staining and ImageJ analysis.
caused a significant decrease in pore size across all groups. With regards to the surface of the scaffolds, on D21
Overall, pre-freezing to −20°C resulted in pore sizes of (Figure 6A), cells displayed higher viability in all groups,
81–163 µm; pre-freezing to −80°C resulted in pore sizes indicating the adequate cytocompatibility of the scaffolds.
of 68–99 µm; and flash-freezing resulted in a significant For example, cell viability in the GelMA group increased
decrease in pore size to 17–50 µm. The most significant from 72.6% to 84.4% between days 1 and 21. The cells on
change in structure was due to flash-freezing the samples the GelMA hydrogels by D21 varied between stretched
before freeze-drying. Previous studies have investigated and rounded morphologies. Scaffolds containing CS/HA
the effect of cooling rate on the porous structure of freeze- and CS/HAMA displayed a more consistent elongated
dried constructs. It has been demonstrated that slower cell morphology. Previously, it was found that HAMA
cooling rates produced porous scaffolds with larger pores. alone resulted in lower cell viability compared to GelMA.
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A slow cooling rate would slow down ice crystal formation, However, in this study, the addition of HA or HAMA did
thereby enabling the growth of larger ice crystals. When not result in any decrease in cell viability. For example, cell
the cooling rate is fast, such as freezing in liquid nitrogen, viability increased from 83.1% to 90.8% for the GelMA/CS/
all the crystallization heat is extracted, and crystallization HAMA group from day 1 to 21. The high cell viability on
starts simultaneously in the entire hydrogel. This results the surface of the scaffolds is probably due to the biological
in the limited formation of ice crystals, and thus, a non- recognition sites of gelatin, including the arginine-glycine-
porous or very small porous structure is formed, which aspartic acid (RGD) sequence, as well as the biological
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was also observed in this study. processes involving HA, such as cell attachment, mitosis,
Small pores can effectively reduce cell migration and proliferation. 15
compared to larger pores. 29,50 However, Stenhamre et al. Figure 6B depicts the cross-section of the scaffolds on
reported that smaller pores, while reducing cell migration, D1, showcasing that cells have infiltrated the scaffold. By
promote cartilage-like formation with larger pores, D21, the cells were still alive in all groups. On the GelMA
resulting in chondrocyte differentiation into the osteogenic scaffolds, the cells grew in random clusters throughout the
pathway. Nonetheless, smaller pores have the benefits of cross-section and were not evenly dispersed, displaying an
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more surface area and cell attachment sites. Additionally, area with a high cell density. For scaffolds containing CS/
it has been found that larger pores require more cells to HA and CS/HAMA, the cells mainly clustered around the
fill the free space within the pore, while smaller pores fill PCL and grew in the direction of the fiber by D21. These
the space more easily. Filling these pores can increase cell results revealed that the 3D-printed meniscal scaffolds
interactions and provide a more 3D microenvironment to could serve as a micro-pattern to guide cells to secrete
promote tissue formation. Further studies revealed that an organized fibrocartilaginous matrix, which is crucial
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regardless of pore size (150–500 µm), cell migration can for the meniscus due to its complex internal structure
be attained with good interconnectivity. Taken together, and biomechanical function. However, cell expansion
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small to medium pores (80–250 µm) are optimal for into the hydrogel matrix was limited due to the hydrogel
cartilage regeneration, i.e., being large enough to promote entrapping the cells. Each channel of the PCL scaffolds
cell infiltration, but small enough for complete pore filling contained a hydrogel material. Once freeze-dried, the

Volume 10 Issue 4 (2024) 504 doi: 10.36922/ijb.3784