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Rueda-Gensini, et al.
that the dispersion of GO would be improved by promoting hydrogel sample. The blends were then mixed thoroughly
protein adsorption before its incorporation into SIS and with a benchtop vortex and incubated for 2 h at 37°C
SISMA hydrogels. To test this hypothesis, GO’s protein under constant agitation. Finally, 100 µL of each sample
adsorption capacity was evaluated by quantifying the after incubation were transferred to a 96-well microplate
difference in protein concentration of cell culture media and the absorbance was measured at 405 nm with the aid
in the presence and absence of dispersed GO. Briefly, of a microplate photometer (Multiskan™ FC, Thermo
GO was dispersed at a concentration of 0.5 mg/mL in a Scientific, Waltham, MA, USA). The reaction product is
DMEM cell culture media solution supplemented with highly chromogenic, meaning that a higher color intensity
10% (v/v) FBS and 1% (v/v) P/S for 2 h at 4°C. The is associated with a higher concentration of primary
resulting solution was then centrifuged at 12,000 rpm for amines. Finally, the reaction efficiency was calculated
5 min to isolate the GO from the medium, and protein with Equation 2.7.1.
concentration in the supernatant was finally quantified
with the Quanti-Pro bicinchoninic acid assay Assay Kit  A SISMA 
(Sigma-Aldrich) and a bovine serum albumin standard %of functionalization = 1 − A   *100 (2.7.1)


curve (Figure S1). The same procedure was conducted, SIS
as a control, for the same supplemented DMEM but in
the absence of GO. Absorbance was measured at 562 nm where A SISMA and A are the absorbance’s of
SIS
with the aid of a microplate photometer (Multiskan™ SISMA and SIS samples, respectively.
FC, Thermo Scientific, Waltham, MA, USA) and 2.8. GO dispersion in SISMA
protein adsorption on the GO sheets was calculated by
implementing Equation 2.5.1. To evaluate if protein adsorption allowed proper GO
dispersion within the hydrogel, serum proteins were
 A  fluorescently labeled with rhodamine B and exposed
Proteinadsorption% = 1 − DMEM GO−   *100 (2.5.1) to GO such that, after dispersion, they can be imaged

A

DMEM
through confocal microscopy. Briefly, the carboxy
terminal of rhodamine B was activated with EDC/NHS
where A is the absorbance measured from the at 37°C for 15 min and subsequently added to a 10%
DMEM-GO
supplemented DMEM in the presence of GO and A DMEM (v/v) solution of FBS. The mixture was left to react
is the absorbance from the supplemented DMEM alone. for 24 h at room temperature (~23°C) under constant
2.6. Hydrogel preparation magnetic stirring. GO was then added at a concentration
of 0.5 mg/mL and, after 2 h of exposure to the labeled
A working solution (pH 8.5) consisting of DMEM proteins, it was thoroughly washed by several cycles of
supplemented with 10% (v/v) FBS, 1% (v/v) P/S, 0.1% centrifugation and resuspension in type II water until the
(w/v) RF, and Tris-HCl 0.1 M was prepared. Next, GO was supernatant showed no traces of rhodamine B. Finally,
dispersed at a 0.5 mg/mL concentration in this working protein-adsorbed GO was resuspended in type II water
solution and left for 12 h at 4°C to promote protein binding. and mixed at a 1:1 volume ratio with a 40 mg/mL SISMA
This was followed by resuspension of lyophilized SISMA solution prepared in 0.02 M acetic acid. The samples were
at 40 mg/mL in a 0.02 M acetic acid solution aided by a imaged at 559 nm with an Olympus FV1000 Confocal
spatula. This mixture was subsequently mixed at a 1:1 Microscope (Tokyo, Japan, Objective 10X) and particle
volume ratio with the working solution containing GO count and area were later analyzed with the ImageJ®
(SISMA-GO). For the hydrogels produced in the absence software. Z-stack reconstruction was also performed by
of GO, the same procedure was performed but with a capturing images at different positions throughout the
working solution without the dispersed nanomaterial. hydrogel depth to assess the spatial distribution of GO
sheets within the hydrogel.
2.7. Quantification of functionalization efficiency

Since MA is conjugated to free-amine residues, 2.9. Rheological evaluation
these were quantified before and after biochemical To assess the rheological response of the developed
modification as an indicator of reaction efficiency. This hydrogels, as well as the influence of photocrosslinking
was achieved through a TNBSA assay according to on their mechanical properties, flow sweep, temperature
the protocol described by Capella-Monsonís et al. . sweep, and time sweep experiments were performed on a
[46]
Briefly, SIS and SISMA hydrogels were diluted in 0.1 M Discovery Series Hybrid Rheometer-1 (TA Instruments,
sodium bicarbonate (pH 8.5) to a final concentration of New Castle, DE, USA) using a parallel plate geometry
0.2% (w/v), and 250 µL of a 0.01% (w/v) picrylsulfonic with a 20 mm gap. Flow sweep experiments of SISMA
acid solution were then added to 500 µL of the diluted and SISMA-GO hydrogels were conducted from 0.01 to

International Journal of Bioprinting (2021)–Volume 7, Issue 3 127