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Multiresponsive Graphene-Oxide Embedded ECM Hydrogel for 3D Bioprinting
2. Materials and methods water and hydrogen peroxide (H O ) were added to the
2
2
blend and the obtained GO was washed successively
2.1. Materials by filtration with polyester fiber, centrifugation, and
Methacrylic acid (MA), EDC, NHS, dimethylformamide resuspension in a solution containing HCl, ethanol and
(DMF), picrylsulfonic acid, RF, formalin solution, TOX8 type I water. The final pellet was resuspended in type II
resazurin-based assay, rhodamine B, sodium bicarbonate, water and lyophilized for 24 h.
and low glucose Dulbecco’s Modified Eagle Medium Adequate GO synthesis was confirmed through
(DMEM) were purchased from Sigma-Aldrich (St. Louis, Raman spectroscopy by assessing the difference between
MO, USA). Sodium dodecyl sulfate was purchased from excitation and emission intensity within a range of 0 and
−1
Santa Cruz Biotechnology (Dallas, TX, USA). Fetal 3000 Raman shift (cm ). A point-wise laser excitation
bovine serum (FBS) and penicillin/streptomycin (P/S) of 532 nm was shone at different locations along the
were purchased from BioWest and hydrogen peroxide synthesized GO sheets to evaluate homogeneity of the
(H O ), sodium chloride (NaCl), sodium hydroxide sample.
2
2
(NaOH), glacial acetic acid, sulfuric acid, phosphoric
acid, potassium permanganate (K MnO ), Tris-HCl, and 2.4. Pre-gel preparation and biochemical
2
4
hydrochloric acid (HCl) were purchased from PanReac modification
AppliChem (Chicago, IL, USA), and graphite flakes Dry SIS powder was solubilized at 4 mg/mL in a solution
from Graphene Supermarket (Ronkonkoma, NY, USA). containing 0.5 M acetic acid and porcine pepsin at a
Propidium iodide, AlexaFluor 488 Phalloidin, and 1 mg/mL concentration. Solubilization was performed
TM
Hoechst 3342 were purchased from Invitrogen (Waltham, under constant vigorous magnetic stirring and at room
MA, USA). temperature (~23°C) for 48 h. The free-amine groups
present in the obtained SIS pre-gels were quantified
2.2. Isolation and decellularization of small by performing the 2,4,6-trinitrobenzene sulfonic acid
intestinal submucosa (SIS) (TNBSA) assay (see section 2.6).
Porcine small intestines were obtained from a local butcher Next, the obtained pre-gel was biochemically
shop and SIS was isolated and decellularized by following modified by covalently linking the C-terminal of MA
the protocol previously reported by Sánchez-Palencia to primary-amine residues present in fibrous structural
and colleagues . Briefly, the jejunum was thoroughly protein backbones of SIS. This was accomplished by
[42]
washed with tap water and the tunica mucosa and serosa adapting the protocol reported by Gaudet et al., which
muscularis layers were mechanically removed to isolate was developed for the biochemical modification of
[44]
the SIS. For decellularization, isolated tissue samples collagen . Briefly, a working solution of MA (in a 1:20
were chemically treated with a proprietary solution molar excess with respect to free amines in SIS), EDC
consisting of H O , sodium hypochlorite, and autoclaved (1:1 molar ratio with MA) and NHS (1:1 molar ratio
2
2
type II water for 15 min under constant agitation at room with MA) was prepared in DMF and heated for 15 min
temperature (~23°C). Next, three successive washes with at 40°C under constant magnetic stirring for activating
autoclaved type II water and one with sterile 1× phosphate- the terminal carboxyl groups of MA. The activated
buffered saline (PBS) were performed to remove cellular solution was then poured into the SIS pre-gel and the
and chemical remnants and for restoring ionic balance, biochemical modification reaction was left for 24 h under
respectively. Finally, the obtained membranes were constant magnetic stirring at 4°C. The resulting modified
dried at room temperature under laminar flow overnight SIS (SISMA) pre-gel was dialyzed against 0.5 M acetic
and subsequently pulverized with the aid of a cryogenic acid for 48 h and subsequently lyophilized for 24 h.
mill (6875 Freezer/Mill, SPEX SamplePrep, Metuchen, The biochemically modified SIS samples were stored at
NJ, USA). Dry SIS powder samples were then stored at −20°C and, if used for bioprinting experiments, sterilized
−20°C until further use. by treatment with ethylene oxide (EtO).

2.3. GO synthesis and characterization 2.5. Quantification of GO protein adsorption
GO synthesis was performed by following the Tour’s capacity
methods with slight modifications . Briefly, a solution Dispersion of GO in acid solutions and biological
[43]
prepared by mixing sulfuric acid and phosphoric acid at buffers is very limited due to the presence of protonated
a 9:1 ratio was carefully poured into a flask containing carboxyl groups and surface charging that induces
graphite flakes and potassium permanganate. The sheet agglomeration . Consequently, its homogeneous
[45]
resulting blend was then heated at 60°C and left for 12 h incorporation into ECM and collagen-based hydrogels is
under constant vigorous magnetic stirring. Next, ice cold a challenging task. To address this issue, we hypothesized

126 International Journal of Bioprinting (2021)–Volume 7, Issue 3