Page 78 - IJB-9-5

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

Table 1. BSA and GMA ratios for modification experiments Table 2. Main components of the as-prepared photoresist
precursors
n lysine /n GMA m BSA n lysine n GMA V GMA Methacrylation
(g) (mmol) (mmol) (μL) degree (%) Sample BSA-GMA (mg, wt%) LAP (mg, wt%) H O (μL)
2
1:1 5 4.45 4.45 577.5 15 R D 125.8 (20 wt%) 3.14 (0.5 wt%) 500
20 52
1:1.5 5 4.45 6.68 865 35 R D 214.3 (30 wt%) 3.62 (0.5 wt%) 500
30 52
1:2 5 4.45 8.90 1155 52 R D 336.2 (40 wt%) 4.2 (0.5 wt%) 500
40 52
R D 336.2 (40 wt%) 4.2 (0.5 wt%) 500
40 35
guidance to the development of functional materials for R D 15 336.2 (40 wt%) 4.2 (0.5 wt%) 500
40
tissue engineering and biomedical fields.
2. Materials and methods the powders with potassium bromide (KBr), while GMA
was coated on the surface of the KBr slice. Proton nuclear
2.1. Materials magnetic resonance ( H-NMR) characterization was used
1
BSA, Hoechst 33342, dialysis membrane, and TESCA to further determine the degree of methacrylation by a
buffer (50 mM TES [C H NO S], 0.36 mM CaCl , pH Bruker DPX FT-NMR spectrometer (9.4 T) at 400 MHz.
6
15
2
6
7.4) were purchased from Solarbio (Beijing, China). BSA (50 mg) and each BSA-GMA were dissolved in 550
GMA and rose bengal (RB) were acquired from Sigma μL Deuterium oxide (D O) and characterized by H-NMR,
1
2
Aldrich (St. Louis, Missouri, MO, USA). Phosphate- respectively. The degree of methacrylation was defined by
buffered solution (PBS; pH = 7.4) was purchased from the percentage of the signal of the methacrylate group at δ
Leagene Biotechnology Co., Ltd, (Beijing, China). Rabbit = 5.7 and δ = 6.1 ppm versus the proton signal generated
articular chondrocytes and rabbit articular chondrocytes by the aromatic amino acids at 6.6–7.4 ppm in BSA-GMA.
complete culture medium were acquired from Procell
Life Science&Technology Co., Ltd (Wuhan, China). %Degree of methacrylation = (A + A ) ⁄A × 100
Mito-Tracker Deep Red and propidium iodide (PI) were δ = 5.7 δ = 6.1 δ = 7.0 (II)
purchased from Invitrogen, Ltd. (UK). LAP was acquired
from TCI Development Co., Ltd (Shanghai, China). 2.4. Preparation of hydrogel precursor solution
2.2. Synthesis of BSA-GMA First, freeze-dried BSA-GMA powder with 52%
BSA (5 g) was dissolved in 25 mL PBS and stirred for half methacrylation degree and 0.5 wt% photoinitiator LAP
an hour at room temperature until completely dissolved. were dissolved in ultrapure water to form precursor
The mature BSA protein with a molecular weight of about solutions of 20 wt% BSA-GMA (52%), 30 wt% BSA-GMA
66.5 kDa contains 583 amino acids, of which 59 are lysines (52%), and 40 wt% BSA-GMA (52%). Then, the BSA-GMA
according to the manual for BSA. powders with 15% and 35% methacrylation degree and
0.5 wt% of photoinitiator LAP were added in ultrapure
n = (m × 59 )/M (I) water to form 40 wt% BSA-GMA (15%) and 40 wt% BSA-
lysine BSA BSA
GMA (35%) precursor solutions. Table 2 shows the main
As shown in Table 1, BSA-GMA was synthesized by adding components of the as-prepared photoresist precursors. The
577.5, 865, and 1155 μL of GMA solution dropwise to the BSA hydrogels are defined by the weight ratio (R) and degree
solution, and the reaction was carried out at room temperature of methacrylation (D) and abbreviated as R D , R D ,
52
20
52
30
for 5 days. The solution was diluted five-fold with PBS and R D , R D , and R D , respectively. The precursor
15
52
40
35
40
40
stirred for 30 min to terminate the reaction. The solution solutions were fully stirred in a dark room and stored in a
was dialyzed with deionized water for 5 days using a 12–14 refrigerator at 4°C.
kDa cut-off dialysis membrane. Finally, the methacrylated
BSA solution was freeze-dried. The freeze-dried BSA-GMA 2.5. TPP microfabrication of BSA-GMA hydrogels
powders were stored at -20°C for further use. In the fabrication experiment, a home-built laser
direct writing optical setup was used to fabricate 3D
2.3. Molecule structure characterization microstructures of BSA-GMA hydrogels (Figure S1
by H-NMR/FT-IR in Supplementary File). A near-infrared Ti: sapphire
1
The powders of BSA and BSA-GMA synthesized with femtosecond laser beam with a central wavelength of
different lysine/GMA molar ratios of 1:1, 1:1.5, and 1:2 were 780 nm, a pulse width of 80 fs, and a repetition rate of
measured by Fourier transform infrared spectrometer (FT- 80 MHz was used. The laser beam was tightly focused into
IR) to evaluate the graft of GMA onto BSA. The samples the photoresist by an oil immersion objective lens (60×,
for FT-IR characterization were prepared by grinding numerical aperture 1.42, Olympus). Before the fabrication
Volume 9 Issue 5 (2023) 70 https://doi.org/10.18063/ijb.752