Page 11 - IJB-9-5

International Journal of Bioprinting 3D printed edible bird’s nests

cells [2,12] . A possible design is to culture salivary gland cells USA) following a previously described method . For
[15]
in one layer-TE-based simulation of the bird’s salivary flow sweep, the rheology study was carried out at room
gland and construct another biomaterials layer to collect temperature. A parallel plate with a diameter of 20 mm
and stabilize the nutritional components from the first and a truncation gap distance of 1,000 μm was applied to
layer. The first layer should facilitate the optimal survival all GelMA samples. To measure the viscosity, these GelMA
and secreting function of salivary gland cells and efficient samples were loaded with steady rate sweeps within a
diffusion of molecules; the second layer should serve as the shear rate range of 0.01–1,000 s . The sweep points were
-1
engineered EBN product for consumption. set with 10 per decade, and the angular frequency ranged
-1
Based on the above assumption, we proposed to from 0.1–100.0 rad s . For temperature sweep, to measure
devise a two-layer model for this “tissue-engineered” the storage modulus and loss modulus, GelMA samples
EBN (TeeBN). For the first “feeding layer,” we synthesized were loaded with steady rate sweeps within a shear rate
-1
gelatin methacrylate (GelMA) and employed 3D printing range of 0.01–1,000 s . The sweep points were set at 10
to encapsulate submandibular gland epithelial cells per decade. To measure the storage modulus (G′) and loss
(SGEC) in GelMA hydrogels. The 3D-printed gel created modulus (G″) in the temperature ranging from 22°C to
a biomimetic niche for the cells to survive—a typical TE 40°C, temperature sweep tests were conducted in the linear
setting—and continuously release nutritional factors. viscoelastic region at a strain of 1%.
Then, we reconstituted food-grade glycan materials 2.4. 3D printing of the feeding layer
according to the carbohydrate proportions of natural EBN The BioScaffolder BS3.2 (GeSiM, Radeberg, Germany)
into the second “receiving layer,” which both provided was used to print the feeding layer. Before cell loading,
the ingredients and, more importantly, stabilized EGF a total of 91 GelMA samples were tested to draw the
released from the feeding layer through carbohydrate- printability window, with the temperature ranging from
growth factor interaction (Figure 1C). After constructing 22°C to 28°C and the pressure from 10 to 130 kPa. GelMA
this TeeBN model, we analyzed its biochemical parameters ink, with 0.25% (w/v) photoinitiator lithium phenyl-2,
in vitro and tested its metabolism in vivo, with emphases 4, 6-trimethyl-benzoyl phosphinate (LAP), was loaded
on how it could retain the essential nutritional factors of into preheated cartridges (26°C) and printed at 4 mm/s
natural EBN, while avoiding heavy metal and microbial through a 27 G, 410 μm conical needle to a cooled
contaminations normally present in the latter. receiving platform (4°C). The final hydrogel scaffold was

2. Materials and methods crosslinked under 405 nm blue light for 10 s. 3D models
for grids and discs were processed with GeSiM Robotics
2.1. Synthesis of methacrylate gelatin (GelMA) version 1.16.0.3892 .
[15]
Gelatin (10 g; Sigma Aldrich, Burlington, USA) was fully For cell-loaded printing, 1 g GelMA was dissolved in
dissolved in phosphate-buffered saline (PBS, 10 mL; Gibco, 10 mL PBS containing 0.25% (w/v) LAP and filtered by
Waltham, USA) at 50°C for 3 h. By referring to a previously 0.22 μm filtration membrane to finally obtain a 10% (w/v)
[13]
reported method , methacrylic anhydride (MA, 8 mL; concentration GelMA as the biomaterial ink. Epithelial Cells
Sigma Aldrich, Burlington, USA) was slowly added (5 × 10 cells/mL) were mixed with 1 mL biomaterial ink.
6
to the gelatin solution in a round-bottom flask. Then, After printing, the medium was soaked for 5 min and then
the methacrylate gelatin samples were dialyzed against replaced with the complete medium culture in the incubator.
deionized water (molecular weight cut-off: 12–14 kDa) at
40°C for 1 week, before being frozen and lyophilized. The 2.5. Cytotoxicity of GelMA scaffolds
synthesized GelMA was verified by H-NMR . Epithelial cells (5 × 10 cells/mL) were cultured with GelMA
1
[14]
3
2.2. Cell culture scaffolds soaking medium in 96-well plates, followed by
Murine submandibular gland epithelial cells (MSGEC) testing in CCK-8 cell viability assay (ApexBio, Houston,
were purchased from YuchiCell (Shanghai, China) and USA) after 1, 3, and 5 days. The working solution consisted
cultured with the DMEM (Gibco, Waltham, USA) with of staining solution and medium at 1:10 (v/v). After a 4-h
10% (v/v) fetal bovine serum (Gibco, Waltham, USA), culture, the absorbance (450 nm) was measured using a
[16]
1% (v/v) GlutaMax (Gibco, Waltham, USA), and 1% (v/v) microplate reader (Molecular Devices, San Jose, USA) .
antibiotics (Gibco, Waltham, USA). Incubator parameters 2.6. Assays to evaluate cell growth in the feeding
were set at 37°C and 5% carbon dioxide.
layer
2.3. Flow and temperature sweep of GelMA Live/Dead assay (Sigma Aldrich, Burlington, USA) was
The modulus of GelMA samples was analyzed by performed to observe the impact of different feeding layers
Discovery HR-2 rheometer (TA Instruments, New Castle, on epithelial cells . The dye was applied 1/1,000 (v/v) of
[16]
Volume 9 Issue 5 (2023) 3 https://doi.org/10.18063/ijb.691