Page 560 - IJB-10-5

International Journal of Bioprinting 3D bioprinting of collagen hydrogels

100.49 ± 3.46 kPa and 6.37 ± 1.88 kPa, respectively (Figure Live/dead cell staining was further employed to assess
4F). The findings emphasized that the CML-scaffold was the cell survival status on the CML-scaffold (Figure 5C). The
significantly stronger mechanically than Col. laser confocal scanning microscopy images displayed that
the original structure of the CML-scaffold remained intact
The mechanical properties of the CML-scaffold were after seven days of culture. HFF-1 cells were uniformly
further investigated through tensile measurements. The distributed on the scaffold and thriving throughout its
maximum tensile stress recorded for the CML-scaffold was entirety, with no dead cells observed on day 7. These results
9.07 kPa, with a corresponding strain of 33.72% (Figure suggest that the CML-scaffold demonstrated outstanding
4G). The elastic modulus was determined to be 45.49 biological activities, promoting cell growth both around
± 2.50 kPa. These findings collectively indicate that the and within the scaffold.
CML-scaffold possesses excellent mechanical strength.
Immunofluorescence staining was utilized to observe
The CML-scaffold was submerged in deionized water the adhesion of HFF-1 cells on the CML-scaffold (Figure
to assess its swelling ratio (SR) at various time points 5D). Fluorescence images captured by the laser confocal
(Figure 4H). The swelling of the CML-scaffold reached microscope revealed that HFF-1 cells adhering to the
equilibrium within 8 hrs, achieving an SR of approximately CML-scaffold were uniformly distributed in a spindle
113%, and no macroscopic alterations were detected in the shape, displaying a complex actin cytoskeleton structure.
scaffold structure. The result highlights that the CML- These observations indicate that the biological scaffold
scaffold exhibited a nearly constant expansion rate and offered by the CML-scaffold substantially promoted the
demonstrated outstanding structural stability. adhesion and spreading of HFF-1 cells.
The degradation characteristics of the freeze-dried The ability of the CML-scaffold to enhance cell migration
CML-scaffold, along with an equivalent mass of Col, were was evaluated by measuring the change in cell scratch area
assessed in a 5 U/mL collagenase solution (Figure 4I). Col (Figure 5E). The inverted fluorescence microscopy results
degraded by 81.06, 95.60, and 97.20% on days 5, 7, and 9, demonstrated that the CML-scaffold markedly enhanced
respectively, and completely degraded by day 11. In contrast, the migration of HFF-1 cells, as quantitatively analyzed,
the CML-scaffold demonstrated a degradation rate of revealing a migration rate of 80.29%, in contrast to the
only 1.91% on day 15, highlighting its robust resistance to blank group, which exhibited a rate of only 26.86% (Figure
degradation. Additionally, the degradation rates of ColMA 5F). Statistical analysis revealed a notable disparity in the
and the CML-scaffold hydrogels post-polymerization were cell migration rate between the two groups.
compared (Figure S3, Supporting Information). Both Reverse-transcription quantitative polymerase chain
hydrogels exhibited progressive degradation over time. reaction (RT-qPCR) was employed to assess the expression
By day 15, the ColMA hydrogel had degraded by 47.08%, of differentiation-related genes in HFF-1 cells cultured on
whereas the CML-scaffold hydrogel displayed a slower the CML-scaffold (Figure 5G). In comparison to the blank
degradation rate of 28.16%, indicating enhanced stability. group, the CML-scaffold markedly enhanced the expression
3.4. In vitro biocompatibility and bioactivity of α-smooth muscle actin (α-SMA), vimentin, Col-I, and
of CML-scaffold collagen type III (Col-III) in HFF-1 cells. These findings
The CCK-8 assay was employed to assess the cytotoxicity of indicated that the CML-scaffold significantly promoted
52
CML-scaffold extract in vitro (Figure 5A). In comparison to the differentiation of fibroblasts into myofibroblasts.
the blank group, the viability of HFF-1 cells cultured in the In summary, the CML-scaffold provided a highly
CML-scaffold extract was 100%, indicating that the CML- bioactive scaffold for HFF-1 cell adhesion, migration,
scaffold exhibited no cytotoxic effects and demonstrated and differentiation.
excellent cytocompatibility. The biological activity of the 3.5. In situ extrusion of CML-scaffold for
CML-scaffold was evaluated through a cell proliferation full-thickness skin regeneration
assay. The proliferation of HFF-1 cells in the CML-scaffold The rat full-thickness skin injury model was employed to
was assessed using the CCK-8 assay on days 1, 3, and 5 assess the reparative effects of the CML-scaffold on full-
(Figure 5B). On day 1 of culture, the relative proliferation thickness skin injuries. The CML-scaffold was precisely 3D
rates for the blank and CML-scaffold groups were 100.00 printed in situ by extrusion onto the site of full-thickness
and 104.98%, respectively. By day 3, the proliferation rates skin defects (Figure 6A). The decision to apply in situ
increased to 104.12 and 111.78%, respectively. On day 5, printing instead of using pre-fabricated printed scaffolds
the levels further rose to 106.73 and 119.56%, respectively, was based on several key advantages. In situ printing
indicating that the CML-scaffold could enhance the enables direct deposition of biomaterials at the injury
proliferation of HFF-1 cells. site, promoting better integration with surrounding tissue

Volume 10 Issue 5 (2024) 552 doi: 10.36922/ijb.4069