Page 288 - IJB-10-5

International Journal of Bioprinting 3D printing of collagen II-scaffolds

Germany). The staining results were visualized using albumin (BSA) for 30 min. The BMSCs were incubated
excitation wavelengths of 496 and 480 nm, and emission with the primary antibody of collagen II (Abcam, USA)
wavelengths of 340 and 516 nm, respectively. at 4°C and subsequently with the secondary antibody
of collagen II (Abcam, USA) at room temperature for
2.5.5. Western blotting 1 h. Finally, the scaffolds were stained with DAPI, and
At day 7 of cell culture, total protein was extracted from images were obtained via a fluorescence microscope
BMSCs. Specifically, BMSCs were lysed in RIPA buffer (Leica, Germany).
(Beyotime, China). The resulting lysate was collected, and
the protein concentration was determined by using the 3. Results
Bradford assay (Sangon Biotech, China). Subsequently,
equal amounts of protein were separated on a 12% SDS- 3.1. Characterization of biopolymers
PAGE gel and transferred onto 0.22 μm polyvinylidene Cellulose nanofiber (CNF) was used as a filler to modify
difluoride (PVDF) membranes (Sigma-Aldrich, USA). In the rheological properties of the collagen II-based
this process, a constant current of 252 mA was applied. hydrogel, and its morphology was studied using TEM.
The membranes were then blocked with 5% skim milk and The fiber diameter was within the nanoscale range (1–
incubated overnight at 4°C with primary antibodies specific 100 nm), and the fiber length was mostly longer than
to focal adhesion kinase (FAK), N-cadherin, Collagen II, 500 nm. Despite 100× dilution of the as-purchased
Aggrecan, and GAPDH. After three washes with Tris- CNF (2 wt%) solution, entanglement was still observed
buffered saline with Tween-20 (TBST; Beyotime, China), the (Figure 1). Furthermore, GPC was conducted to
membranes were incubated with horseradish peroxidase- determine the molecular weight and distribution of
conjugated secondary antibodies at 25°C for 1.5 h. Finally, collagen II and alginate (Table 1).
the membranes were washed again with TBST, and the
immunoreactive bands were visualized using an enhanced 3.2. Rheological properties of collagen II-containing
chemiluminescence detection kit (Beyotime, China). hydrogel inks
In our preparation of the hydrogel ink, it was determined that
2.5.6. Immunofluorescence staining 3% (w/v) collagen II is the highest feasible concentration,
Bone MSCs (BMSCs) cultured on the scaffold were potentially reaching its solubility limit. According to
treated with 4% paraformaldehyde solution for 15 Figure 2, all the hydrogel inks exhibited a shear-thinning
min, permeabilized with 0.1% Triton X-100 solution property, and the addition of CNF increased the viscosity
in PBS for 10 min, and blocked with 5% bovine serum and shear stress of the inks.

Figure 1. Transmission electron microscope (TEM) images of cellulose nanofiber at a magnification of (A) ×2700, (B) ×5300, and (C) ×13,500.

Table 1. Molecular weight of collagen II and alginate determined from gel permeation chromatography (GPC)

Substance M (Da) M (Da) M (Da) Polydispersity
n w z
Collagen II 25,774 533,724 1,685,898 20.71
Alginate 26,281 527,004 1,632,056 20.05
Abbreviations: M , number average; M , weight average; M , Z-average.
n w z

Volume 10 Issue 5 (2024) 280 doi: 10.36922/ijb.3371