Page 404 - IJB-9-2

International Journal of Bioprinting 3D-printed skin substitute accelerates wound healing in vivo

2.2. Preparation of dECM-GelMA-HAMA precursor heating in 55°C water bath, and filtered by 0.22 μm filter

2.2.1. Adipose tissue decellularization to remove bacteria. HAMA lyophilized powder (SunP
Biotech, Beijing, China) was dissolved in DMEM/F12
Adipose tissue was collected from patients who underwent medium and stirred overnight at room temperature to
thigh or abdominal liposuction in Department of Burn and ensure complete dissolution, followed by pasteurization
Plastic Surgery, the Fourth Medical Centre, Chinese People’s (70°C for 30 min and 4°C for 10 min, repeated 3 times).
Liberation Army General Hospital. Consent of the patients The dECM-GelMA-HAMA precursor composed of
was obtained. Decellularization of adipose tissue was carried 1.125% (w/v) dECM, 7.5% (w/v) GelMA and 1% (w/v)
out by means of enzymatic digestion and organic solvent HAMA was prepared by mixing GelMA and HAMA
[8]
extraction , which is called Flynn’s method. After the first polymer solution with dECM pre-gel. The photoinitiator
trypsin digestion, organic solvent extraction was changed (2.5% lithium phenyl-2,4,6-trimethylbenzoylphosphinate
every 12 h for 48 h to maximize fat content removal. Briefly, or 2.5% LAP) was added, and the hADSCs suspension
the decellularization protocol encompasses repeated freezing obtained, as described in section 2.1, was mixed with
and thawing of adipose tissue for 3 times, centrifugation, precursor at the ratio of 1: 9 to obtain bioink in which the
0.25% trypsin-EDTA digestion, isopropanol extraction, final concentration of hADSCs is 1.0 × 10 cells/mL and
7
trypsin digestion, 1000 U/ml nuclease (Merck, Germany) and photoinitiator is 0.25% (v/v).
type Ⅱ lipase (Solarbio, China) digestion, and isopropanol
extraction until adipose tissue dECM was obtained. All 2.3. Properties of dECM-GelMA-HAMA precursor
the decellularization solutions were supplemented with 2.3.1. Rheological analysis
phenylmethylsulfonyl fluorid (PMSF; Sigma, USA) and 1% The storage (G’) and loss modulus (G’’) of dECM-GelMA-
(v/v) penicillin-streptomycin. Adipose tissue dECM was HAMA precursor obtained in section 2.2.4 were
lyophilized with a vacuum freeze-drier for 48 h. evaluated using a Discovery HR-20 Hybrid Rheometer

2.2.2. Evaluation of the effect of decellularization (TA Instruments Ltd., New Castle, DE, USA). Precursor
was placed on a 40-mm diameter parallel plate separated
To evaluate the effect of decellularization, 4’,6-diamidino- by a 1-mm gap. The strain was fixed at 2%, the frequency
2-phenylindole (DAPI) staining was used to assess the was 1 HZ, and the temperature was changed from 0°C
presence of nuclei. The lyophilized dECM was embedded from 35°C, rising at the rate of 3°C/min.
in paraffin, sectioned, stained with DAPI, and observed
by fluorescence microscope. A small amount of adipose 2.3.2. Scanning electron microscope (SEM) imaging
tissue and dECM was used for DNA quantification. The To confirm the morphological properties and pore
residual DNA of adipose tissue and dECM was extracted structure of the photo-crosslinked dECM-GelMA-
by a genomic DNA extraction kit (Tiangen, Beijing, China) HAMA composite hydrogel, the dECM-GelMA-HAMA
following the instruction, and its content was measured by precursor containing 0.25% (v/v) photoinitiator was
NanoDrop (Thermo scientific, USA). exposed to 405 nm UV for 10 seconds to crosslink. The
2.2.3. Preparation of adipose tissue dECM pre-gel composite hydrogel was fixed, dehydrated and dried,
cut with a scalpel to expose the internal structure and
The lyophilized adipose tissue dECM was digested for placed on the conductive adhesive with the cross-section
solubilization. Brief steps were as follows: adipose tissue facing up, sputter-coated with iridium and observed with
dECM was ground into powder and sieved and was added scanning electron microscope (SEM; S-4800, HITACHI,
to 0.5 mol/L hydrochloric acid together with pepsin at the Tokyo, Japan) to calculate porosity and pore size of the
ratio of 10 mg dECM: 1 mg pepsin with continuous stirring composite hydrogel with ImageJ. Images were acquired at
for 72 h at room temperature to make sure dECM is fully the accelerating voltage of 15 kV and operating distance is
digested. The pH of digestion solution was adjusted with 10 mm.
10 mol/L sodium hydroxide, and the ion concentration
was adjusted with 10× DMEM/F12 to physiological state 2.4. 3D-bioprinted dECM-GelMA-HAMA skin
on ice. Following the steps above, the dECM pre-gel substitute loaded with hADSCs
solution was obtained. The dECM-GelMA-HAMA bioink loaded with hADSCs
obtained in section 2.2.4 was transferred to bioink
2.2.4. Preparation of dECM-GelMA-HAMA bioink container to print dECM-GelMA-HAMA scaffold loaded
loaded with hADSCs
with hADSCs layer by layer using a commercial extrusion-
GelMA lyophilized powder (SunP Biotech, Beijing, China) based 3D printer (Envision TEC, Germany). The
was completely dissolved in DMEM/F12 medium through parameters were as follows: print nozzle: 27 G with inner

Volume 9 Issue 2 (2023) 396 https://doi.org/10.18063/ijb.v9i2.674