Page 40 - IJB-9-4

International Journal of Bioprinting 3D-Bioprinted human lipoaspirate-derived cell-laden skin constructs

sample was pulverized in a freeze-grinding machine. The respectively. A bioink solution of 7.5% w/v GelMA + 1% w/v
adECM powder was then digested with 0.5 M acetic acid HAMA without adECM was prepared. After the bioink
and 10% pepsin (Sigma-Aldrich, USA) relative to the solutions were pasteurized, a 10% volume concentration
adECM weight for 72 h. After the powder was completely of the photoinitiator lithium phenyl-2,4,6-trimethyl
dissolved, the pH of the solution was adjusted to 7.4 with benzoyl phosphinate was added. Before bioprinting,
10 M NaOH, while the temperature was kept at <10°C to ADSCs were detached using 0.25% trypsin/0.1% EDTA.
avoid adECM gelation. The pH-adjusted adECM hydrogel After centrifugation, cells were suspended in phosphate-
was stored at 4°C. buffered saline (PBS) and then mixed with the bioink at
a 1:9 ratio. The final working concentration of cells was
2.2. Evaluation of adipose-derived 1.0 × 10 cells/mL in the cell-laden bioink. 3D bioprinting
7
decellularized ECM was performed as soon as the bioink with all components
To evaluate the decellularization efficiency, residual DNA was prepared.
within the adECM was extracted using a genomic DNA
extraction kit (Tiangen, China) and measured using the 2.5. Rheological characterization of bioinks
NanoDrop system (Thermo, USA). The DNA quantity The rheological properties of the two acellular bioinks with
was normalized to the initial dry weight of the tissue. and without adECM were analyzed using a HR-20 hybrid
Sircol collagen and Blyscan sGAG assay kits (Biocolor, rheometer (TA Instruments, USA) equipped with a 40-mm
UK) were used to evaluate the collagen and sulfated diameter parallel-plate geometry and a gap size of 1 mm.
glycosaminoglycan (sGAG) content, respectively, of The storage (G′) and loss modulus (G″) were recorded at a
decellularized samples. Hematoxylin and eosin (H&E) rate of 3°C/min from 0°C to 35°C under a fixed frequency
staining was performed to visualize any remaining nuclei. of 1 Hz and strain of 2%. The viscosity was measured in
An Oil Red O staining kit (Solebo, China) was used to stain two aspects. First, the effect of temperature on the viscosity
residual lipids in the adECM. was observed at a fixed shear rate of 1 1/s from 0°C to 30°C.
Then, the effect of shear rate on viscosity was recorded for
2.3. Culture and identification of ADSCs a shear rate change from 0.1 to 100 1/s at 17°C. A time-
ADSCs were obtained from the adipose tissue of the patient sweep oscillatory test with a fixed frequency of 1 Hz, strain
undergoing liposuction as mentioned in section 2.1. of 2%, and temperature of 25°C was used to record the
Isolation and culture of ADSCs were performed as modulus changes of the samples before and after 10 s of
previously described . Liposuction was digested with UV irradiation with a 405-nm UV lamp (CREE, China).
[31]
0.1% collagenase type I (Sigma-Aldrich, USA) for 60 min
at 37°C. Samples were then filtered through a 200- 2.6. Swelling of photocrosslinked bioinks
mesh filter. The filtrate was centrifuged at 1200 ×g for Photocrosslinked samples of the two acellular bioinks
5 min. Pelleted cells were washed and resuspended in with and without adECM were immersed in PBS at 37°C
L-DMEM (HyClone, USA) supplemented with 10% fetal for the following time intervals: 1, 6, 12, and 24 h. At each
bovine serum (FBS) and 1% penicillin–streptomycin– timepoint, excess PBS was drained from scaffold surfaces,
amphotericin in a humidified atmosphere of 5% CO at and the scaffold weight was then measured and recorded as
2
37°C. Cells from the third to fifth passages were used for W . Scaffolds were lyophilized for 1 week, and the weight of
1
experiments. Commercial kits (Cyagen, China) were used each after lyophilization was recorded as W . The swelling
0
to examine the adipogenic, osteogenic, and chondrogenic ratio (SR) was calculated using Equation I:
differentiation abilities of the ADSCs following the SR = (W − W )/W (I)
manufacturer’s protocols. Cells were stained with Oil Red 1 0 0
O, Alizarin red, and Alcian blue to assess adipogenic, 2.7. Degradation of photocrosslinked bioink
osteogenic, and chondrogenic differentiation, respectively. scaffolds
Flow cytometry (BD Biosciences, USA) was used to detect Scaffolds prepared from the two acellular bioinks were
the expression of cell surface markers, such as CD31, lyophilized, and initial masses were recorded as W . Dried
0
CD34, CD45, CD73, and CD90. scaffolds were rehydrated in PBS for 24 h and subsequently
transferred to PBS containing 1.5 U/mL collagenase II for
2.4. Bioink formulation and cell encapsulation incubation with shaking at 37°C. Samples were removed
To improve adECM hydrogel formability for 3D printing, at different time points for lyophilization, and the weights
GelMA and HAMA (SunP Biotech, China) were used of dried samples were recorded as W . The percent of
as composite components of the prepared bioink herein remaining mass (RM) after degradation was calculated
t
based on their photocrosslinked properties. The final using Equation II:
concentrations of adECM, GelMA, and HAMA in the
bioink formulation were 1.125%, 7.5%, and 1% w/v, RM(%) = W /W × 100% (II)
0
t
Volume 9 Issue 4 (2023) 32 https://doi.org/10.18063/ijb.718