Page 486 - IJB-10-4

International Journal of Bioprinting Embedded bioprinting of cartilage

2. Materials and methods Before the preparation of the support bath, the
supernatant of G/A microparticles was removed by
2.1. Materials centrifugation. The microparticles were then mixed with
Materials for the synthesis of OAlg and Gel-CDH were the OAlg solution (16.5% w/v in Dulbecco’s phosphate-
purchased from Sigma-Aldrich (United States of America buffered saline [DPBS]) to obtain the granular support bath.
[USA]) unless indicated otherwise. All reagents were used
as received without further purification. 2.4. Rheological measurements
Rheological measurements were performed using a
2.2. Synthesis of carbonyl rotational rheometer (MCR 302; Anton Paar, USA)
hydrazide-modified gelatin with a parallel plate rotator of 25 mm diameter. The
To synthesize Gel-CDH, 3 g of gelatin was dissolved in 300 rotational tests were performed to determine the shear-
mL of deionized (DI) water, and 2.2 g of carbohydrazide thinning behavior of support baths, including 80% G/A
(Aladdin, China) was added during magnetic stirring. microparticles + 20% OAlg, 70% G/A microparticles +
Next, 450 mg of N-(3-dimethylaminopropyl)-N’- 30% OAlg, 60% G/A microparticles + 40% OAlg, 50% G/A
ethylcarbodiimide hydrochloride (in 10 mL DI water) microparticles + 50% OAlg, and 40% G/A microparticles
and 450 mg of 1-hydroxybenzotriazole hydrate (in 10 mL + 60% OAlg (referred to as 80%, 70%, 60%, 50%, and 40%
dimethyl sulfoxide; Aladdin, China) were added dropwise support bath concentration hereafter, respectively), at a
during magnetic stirring. The pH of the solution was shear rate of 0.1–100 s . The storage modulus (G’) and loss
−1
adjusted to 5.25, and the reaction was carried out at room modulus (G’’) were measured to evaluate the viscous and
temperature overnight. The solution was then dialyzed in elastic properties of support baths via amplitude tests at
0.3 M sodium chloride (NaCl) solution, 25% v/v ethanol a constant frequency of 1 Hz, a strain of 0.01–100%, and
solution, and DI water for 2, 1, and 4 days, respectively, a constant temperature of 23°C. Thixotropy curves for
using a dialysis membrane with a 7 kDa molecular weight different support baths were obtained by a three-stage test
cutoff (MWCO; Solarbio, China). The pure product was protocol: (i) low shear rate (0.01 s ) for 1 min, (ii) high
−1
then attained by freeze-drying.
shear rate (10 s ) for 10 s, and (iii) low shear rate (0.01 s )
-1
−1
2.3. Preparation of support bath for 1 min. The rheological properties of the 12% w/v Gel-
The support bath was composed of microparticles CDH solution (in DPBS) were also evaluated. Rotational
suspended in an OAlg solution. To synthesize OAlg, 9 g of and oscillatory tests were applied for temperatures of 10–
sodium alginate was dissolved in 450 mL of DI water (2%, 40°C to explore the temperature-dependent viscosity and
w/v) and stirred overnight. Sodium periodate (3 g; Aladdin, dynamic modulus of a 12% Gel-CDH solution.
China) that was dissolved in 30 mL of DI water (10%, w/v)
was added to the solution and stirred magnetically in a dark 2.5. Computational fluid dynamics simulation
environment for 24 h at room temperature. Thereafter, 6 Computational fluid dynamics (CFD) simulation was
mL of ethylene glycol was added to terminate the above performed using ANSYS 2021 software (Ansys, Inc.,
oxidation reaction, and the solution was stirred for another USA). A 3D geometric model was constructed and meshed
hour. Approximately 3 g of NaCl was dissolved in the final according to the internal dimensions of the nozzle. The
solution for 15 min. The solution was then dialyzed in DI fluid model was set as an incompressible fluid, disregarding
water for 3 days using a dialysis membrane with MWCO 7 the pressure loss of the flow in the nozzle. The Reynolds
of kDa and lyophilized. number was calculated based on the following equation:

To prepare G/A microparticles, 1 g of alginate was
n 2
dissolved in 45 mL of DI water and mixed with 5 g of R = ρdv −n (I)
gelatin. Thereafter, 1 g of transglutaminase (TG; Yiming e K
Biological Technology, China) was dissolved in 5 mL of DI
water and added to the above solution. The solution was where ρ is the density of bioink, d is the nozzle diameter,
mixed thoroughly and kept overnight in a 37°C water bath v is the flow rate of bioink, K is the coefficient of viscosity,
to ensure crosslinking. The semi-crosslinked G/A hydrogel and n is the flow behavior index. The n and K values
was then mechanically ground into microgel slurry by a were obtained by fitting the rheological results with the
blender (IKA, Germany). The slurry was loaded into 50 Herschel–Bulkley (H–B) model. The estimated Reynolds
mL conical tubes and centrifuged at 7600 × g for 1 min. number indicated that the flow pattern of the fluid during
The supernatant was replaced with DI water, followed by the extrusion process was laminar. In addition, the energy
the resuspension of G/A microparticles and storage at 4 °C equation was used due to the temperature sensitivity of
for further use. Gelatin microparticles were also prepared bioinks. The temperature of the nozzle hub was set to 37°C
without the addition of alginate for comparison. and the rest regions were set to room temperature (26°C).

Volume 10 Issue 4 (2024) 478 doi: 10.36922/ijb.3520