Page 79 - IJB-9-5

International Journal of Bioprinting Biocompatible BSA-GMA and TPP of 3D hydrogels with free radical type I photoinitiator

processing, the BSA-GMA precursor solution was dropped a CCD DS-Ri2 (Nikon, Japan) and a 50× objective lens
between two coverslips placed on an XYZ positioning (N.A. = 0.8). pH response experiments were performed
stage. The photoresist was scanned by a femtosecond laser with pH 2, pH 5, PBS (pH 7.4), and pH 11 solutions, in
beam in 3D by a computer-controlled XYZ motorized which the solutions of different pH were prepared with
stage (Physik Instrumente). After the fabrication, the concentrated HCl and NaOH.
unpolymerized BSA-GMA solution was washed with
deionized water, and the desired 3D microstructure 2.9. Evaluation of cell viability in BSA-GMA
can be achieved. Scanning electron microscope (SEM) hydrogels
images were characterized by a field emission scanning 3D microscaffolds of R D , R D , R D , R D , and
52
30
40
52
40
52
20
35
electron microscope (FESEM, Hitachi S-4800). Confocal R D were prepared in each coverslip by the TPP technique.
15
40
fluorescence images were recorded by a laser scanning Before cell culture, the cell scaffolds were sterilized with
confocal fluorescence microscope (A1 MP, Nikon). 75% ethanol and ultraviolet (UV) light for 1 h, respectively.
Rabbit articular chondrocytes were cocultured with five cell
2.6. Optical characterization of precursors and scaffolds using chondrocyte-specific medium, and incubated
microstructures of BSA and BSA-GMA in a humidified incubator at 37°C and 5% CO for 72 h. The
2
UV-Vis absorption spectra were characterized medium was changed every other day. The mitochondria
by a Shimadzu UV-2550 spectrophotometer. The of living cells were stained with Mito-Tracker Deep Red
concentration of initiator in the initiator solution was for 20 min, and then, the nuclei of living cells were stained
8.55 × 10 g mL . The BSA-GMA aqueous solution with Hoechst 33342 for 10 min. Dead cell nuclei were then
-1
-3
was prepared from BSA-GMA powder with 52% degree stained with PI for 10 min. The fluorescence images were
of methacrylation and 40 wt% concentration without observed using confocal fluorescence microscope with a
initiator as a control, as well as the solutions of R D , 20× objective lens. The excitation wavelengths were 640,
52
20
R D , R D , R D , and R D containing 0.5 wt% 561, 488, and 405 nm, respectively.
40
52
15
30
35
52
40
40
initiators. Infrared spectra of GMA, precursor solution,
and microstructure of BSA+RB, as well as precursor 3. Results and discussion
solution and microstructure of BSA-GMA were
performed with a Fourier transform infrared micro- In this work, GMA was used to modify BSA to obtain a
spectrometer (Vertex 70 Micro and Hyperion 1000-2000) series of BSA-GMA materials, which can not only be two-
in the range of 600 to 4000 cm . Raman spectra and photon polymerized using water-soluble radical type I
-1
images were measured by a Raman-11 microscopy using photoinitiators without depleting the amino acid groups
a 532-nm laser as the excitation source in line scanning of the protein, but can also be adjusted according to the
mode. degree of methacrylation to tune the TPP capability.
Moreover, the polymerized BSA-GMA hydrogel structure
2.7. Zeta potential and isoelectric point of BSA and exhibited autofluorescence, pH response, and excellent
BSA-GMA with different methacrylation degrees biocompatibility.
The electrical properties were characterized using a
Zetasizer (Nano ZS90, Malvern, UK) under different 3.1. BSA-GMA preparation and characterization
pH conditions. Sodium phosphate dibasic-Citric acid BSA has low toxicity, high biocompatibility, and good
(Na HPO -CA) buffer solutions were prepared at pH flexibility, and can be easily polymerized into hydrogels,
4
2
2.66, 3.15, 3.45, 3.79, 4.07, 4.52, 4.96, 5.22, 6.09, 6.97, and showing a wide range of applications in various fields. BSA
8.16, respectively. BSA and BSA-GMA were dissolved in consumes a large number of amino acid residues via TPP
Na HPO -CA buffer solutions at different pH values at a based on a dye-amine system. Many of these amino acid
2
4
concentration of 10 μg mL , and the zeta potential values residues are important for cells, which are essential for cell
-1
of various samples were measured. The isoelectric point is attachment to protein scaffolds. Thus, we modified BSA by
the pH value at a zeta potential of 0 mV. The isoelectric GMA so that the TPP reaction only occurs at the double
point of the material is obtained by a linear fitting to two bond on the GMA chain without consuming amino acid
pH values across 0 mV. groups with type I initiators. This overcomes the limitation
2.8. pH response experiment of consuming amino acids using free radical type II
First, 7 × 7 × 2 μm sized hydrogel cubes were polymerized initiators in TPP of BSA (Figure 1). The BSA-GMA was
3
by the TPP technique. Afterward, 200 μL of different pH synthesized by replacing the primary amines of BSA with
solutions were dropped onto coverslips containing the GMA (Figure 1A). GMA reacts with the primary amine
hydrogel structures at room temperature, and the pH (-NH ) of the lysine residue of BSA via an epoxide ring-
2
response process was recorded under a microscope with opening mechanism without acidic by-products . GMA
[52]
Volume 9 Issue 5 (2023) 71 https://doi.org/10.18063/ijb.752