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Chen, et al.
and modification of printing materials coupled resulting solution was transferred to a plastic mold
with in situ post-printing functionalization and and exposed to ultraviolet (UV) light for 5 min. The
hybridization in reactive viscoplastic matrices. gel piece was subsequently physically crosslinked
in a 0.153 M CaCl solution for 3 h and dialyzed
2
2 Materials and methods in distilled water for 24 h. To compare composite
2.1 Materials hydrogels through in situ precipitation with those
through physically mixing with CaP nanoparticles,
HAc sodium salt from Streptococcus equi CaP nanoparticles were prepared by mixing CaCl
2
(molecular weight ≈ 1.5~1.8 MDa), phosphate- solution (0.153 M) and (NH ) HPO 0.092 M).
4 (
4 2
buffered saline (PBS), dimethylformamide The CaP precipitate was then centrifuged and
(DMF), GM, tetrabutylammonium bromide, lyophilized. GM-HAc (1% wt/v) was mixed
triethylamine, acetone, alginic acid sodium salt with Alg (0.125% wt/v), CaP (0.34% wt/v), NVP
from brown algae, N-vinylpyrrolidinone (NVP), (5% v/v), and Irgacure 2959 (1% wt/v). Then,
Irgacure 2959 (2-Hydroxy-4′-(2-hydroxyethoxy) 1 mL of the resulting solution was transferred
-2-methylpropiophenone), calcium chloride (CaCl ), to a plastic mold and exposed to UV light for
2
ammonium phosphate dibasic ((NH ) HPO ), and 5 min. The gel piece was subsequently physically
4 2
4
gelatin from bovine skin (Type B) was purchased crosslinked in a 0.153 M CaCl solution for 3 h
2
from Sigma-Aldrich and used without further and dialyzed in distilled water for 24 h.
purification. 2.4 Preparation of 3D printed hydrogel scaffolds
2.2 Preparation of GM-HAc The gelatin bath was prepared using a previously
[17]
GM-HAc was synthesized using a previously developed protocol . In brief, gelatin (5% wt/v)
developed protocol . HAc (1% wt/v) was dissolved was dissolved in a 0.153 M CaCl solution at 40°C.
[9]
2
in a mixture of PBS and DMF at the volume The solution was then gelled at 4°C. Subsequently,
ratio of 1:1. Then, triethylamine (4.4% v/v), GM 5 mL of the gelatin gel and 15 mL of the 0.153 M
(4.4% v/v), and tetrabutylammonium bromide CaCl solution was homogenized at 10,000 rpm for
2
(4.4% wt/v) were added in sequence. After stirring 1 min. The mixture was centrifuged at 4000 rpm
overnight at room temperature, GM-HAc was for 2 min, and the supernatant was removed to
precipitated with acetone and dissolved in distilled obtain a gelatin slurry support bath.
For 3D printing of HAc-Alg scaffolds, GM-
water to remove excess reactants. The solution was HAc (4% wt/v) was mixed with Alg (0.5% wt/v),
dialyzed in distilled water for 2 days, lyophilized, NVP (10% v/v), and Irgacure 2959 (2% wt/v) to
and stored at 4°C. Through the characterization prepare the ink for 3D printing. The HAc-Alg pure
of GM-HAc using nuclear magnetic resonance hydrogel scaffold was then printed in the gelatin
(Bruker Avance II 300 MHz, Bruker, Germany), slurry support bath at a feed rate of 5 mm/s and air
the degree of methacrylation was found to be pressure of 2.5 bar using a regenHU 3D discovery
~15% (Supplementary Figure 12). printer. The scaffold was then exposed to UV
2.3 Preparation of bulk hydrogel specimens light for 5 min and incubated at 37°C to melt and
through molding remove the support bath.
For HAc-Alg/CaP composite hydrogel
For the fabrication of HAc-Alg and HAc-Alg/CaP scaffolds, the hydrogel inks were prepared by
bulk specimens, GM-HAc (1% wt/v) was mixed mixing GM-HAc (4% wt/v) with Alg (0.5% wt/v),
with Alg (0.125% wt/v), NVP (NVP; 5% v/v), NVP (10% v/v), and Irgacure 2959 (2% wt/v) with
and Irgacure 2959 (1% wt/v) with or without (NH ) HPO 0.092 M) and then printed in the
4 (
4 2
(NH ) HPO , varying the amount of (NH ) HPO gelatin slurry support bath at a feed rate of 5 mm/s
4 2
4 2
4
4
from 0.046 M up to 0.092 M, to yield CaP content and air pressure of 2.5 bar using the regenHU 3D
ranging from 15 to 30 wt%. Then, 1 mL of the discovery printer. The scaffold was then exposed
International Journal of Bioprinting (2020)–Volume 6, Issue 2 31
