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Mishbak, et al.
Alginate is a suitable material for bioprinting . It is a After the chemical modification reaction, the polymeric
[12]
natural water-soluble linear polysaccharide derived from solution was precipitated and totally mixed in 100 mL of
alginic acid, extracted from several species of brown ethanol (100% ethanol) and dried in an oven overnight
algae, such as Laminaria hyperborea, Ascophyllum at 50°C. The precipitate polymer was dissolved in
nodosum, and Macrocystis pyrifera [13-15] . Its structure distilled water (diH O), loaded in the dialysis tubes
2
contains 1,4-linked β-D-mannuronic (M) and α-L- membranes (SnakeSkin Dialysis Tubing from Thermo
guluronic (G) acid residues (Figure 1), arranged in a Fisher Scientific, UK), sealing both sides and dialyzed
non-regular and block-wise fashion along the chain [16-18] . the solution against NaCl for 7 days with periodic water
Alginate shows good biocompatibility, low cytotoxicity, changes every day. The solution was freeze at −80°C and
and high-water content (high swelling ratio), mimicking the polymer recovered by lyophilization.
the structure of the natural extracellular matrix [19-23] .
These properties make alginate a suitable material for 2.2 Characterization of Pre-polymerized
wound dressings, drug delivery systems, and soft tissue Methacrylate Alginate
engineering applications .
[24]
This paper investigates the preparation of alginate-based 2.2.1 Nuclear magnetic resonance (NMR)
systems for ultraviolet (UV) bioprinting applications. The The chemical structure of functionalized alginate was
material is characterized both before and after the curing assessed through HNMR spectroscopy, using the B400
1
process, and the effect of functionalization time on the Bruker Avance III 400 MHz (Billerica, Massachusetts,
rheological, mechanical, morphological, swelling, and USA). Polymeric materials were dissolved in deuterium
degradation properties was investigated. The effect of oxide (Sigma-Aldrich, UK), transferred to NMR tubes
photoinitiator concentration in terms of rheological and and the spectra acquired with 128 scans.
mechanical properties is also assessed and discussed.
The biological characteristics are not reported in 2.2.2 Rheological characterization
this manuscript, but preliminary results with human The rheological tests were performed to characterize
chondrocytes show that the materials considered here do the viscoelastic behavior of both pre-polymerized and
not present any cytotoxicity and allow cell attachment polymerized alginate. The rheological assessment of
and proliferation . pre-polymerized alginate systems was carried out using
[26]
the DHR2 TA Instrument (USA). Samples were placed
2. Materials and Methods between two parallel plates and two different tests

2.1 Synthesis of Methacrylate Alginate (rotational and oscillation) were considered. Rotational
tests were performed to evaluate the viscosity and
Photocurable alginates were prepared through a material strength. In these tests it is assumed that the
functionalization mechanism with methacrylate material flows by applying stress, being the response
anhydride (MA) . Briefly, sodium alginate powder 1%, measured alongside time (temperature was not
[27]
2%, and 3% (w/v) (Sigma-Aldrich, UK) was dissolved considered in this work). Controlled stress was applied,
in Dulbecco’s phosphate-buffered saline (Sigma-Aldrich, and the resulting movement measured. Oscillation
UK) and then mixed with MA (Sigma-Aldrich, UK) at tests were considered to evaluate the viscoelastic
15 mL MA/g of alginate under vigorous stirring. The pH behavior of the material and dynamic moduli. The
of the solution was kept around 7.4–8.0 during the reaction viscoelastic behavior was characterized by measuring
time by adding 5M of NaOH. Maintaining a higher pH the energy stored (storage modulus, Gʹ) in the material
during the reaction is crucial since higher pH enhances during shearing and the energy subsequently lost (loss
the reaction among the amine and hydroxyl groups, which modulus, Gʺ). Shear strain was controlled by varying
lead to a higher degree of modification . Two different the oscillation amplitude.
[28]
reaction times (8 and 24 h) were used to assess the effect To assess the rheological changes during the
of the reaction time on the degree of functionalization. photopolymerization process, the rheological tests were
carried out using the Bohlin Gemini system (Malvern
Instruments) equipped with the OmniCure S1000 light
®
source irradiating in the range of 254–450 nm wavelength,
and oscillation tests were considered. The light intensity
was 10 mW/cm .
2
The rheological behavior of the materials is described
by the following equation:
Figure 1. Alginate showing a linkage between the mannuronic and n
 
guluronic acid .    (1)
[25]
International Journal of Bioprinting (2019)–Volume 5, Issue 2 13