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Zheng, et al.
2.3. Decellularization evaluation by the room temperature CD. The scanning speed was
60 nm, scanning band was 190 – 260 nm. The average
The residual DNA content of the dECMs was measured of three scan results after subtracting the baseline is
to evaluate the degree of decellularization. The total the ellipticity. According to the results of CD at room
DNA was extracted using TIANamp genomic DNA temperature, the variable temperature CD analysis was
kit (Tiangen, Beijing, China). The DNA concentration
was quantified by NanoDrop 2000 spectrophotometry performed. The detection wavelength was 199 nm, the
(Thermo Scientific, USA) and the size of the remaining starting temperature was 30°C, the ending temperature
was 90°C, and the heating rate was 1°C/min. The thermal
DNA fragments was verified by gel electrophoresis. denaturation curve was, then, analyzed by a fitting
Fresh ovarian tissues and the dECMs were
embedded in paraffin. The cell residues in the tissues equation model (a sigmoid curve) to obtain the thermal
were detected using hematoxylin and eosin (H&E) denaturation temperature (Tm) value.
staining and 4,6-diamidino-2-phenylindole (DAPI) (3) Rheological characterization
staining. The dECMs components such as collagen and
proteoglycan were assessed by Masson staining and To assess the viscosity and the strain sweep of the bioink
toluidine blue (TB) staining. The presence of proteins (before and after cross-linking of calcium chloride) and the
or peptides was detected by SDS–polyacrylamide gel dECM solution (pH 3.2 – 3.5), we conducted rheological
electrophoresis (SDS-PAGE). Routine electrophoresis, investigation on a rotation rheometer (Malvern Kinexus
dyeing, and discoloration were performed in turn. Ultra+) at 25°C. Amplitude sweep (0.01 – 100 strain,
The microstructure of the dECMs was observed using 10% rad/s) was performed to assess the strain dependent
scanning electron microscopy (SEM; JSM-5600LV, storage modulus (G’) and loss modulus (G’’).
JEOL).
(4) Biocompatibility of the dECMs bioink in vivo
2.4. Preparation of dECM-based bioink Twelve 8-week-old female Kunming mice were

The dECMs were pulverized using a small grinder with subcutaneously injected with 400 µl bioink on the back of
th
nd
st
the help of liquid nitrogen. 100 mg dECM powder was each mouse. The injections were taken at the 1 , 2 , 4 ,
th
taken, 3 ml hydrochloric acid solution (pH 2.0) and and 9 week for H&E staining and immunohistochemistry
60 mg pepsin were added to the dECM powder and (IHC) Staining (CD45, an inflammatory marker).
digested at 37°C for 24 h. The dECM solution pH was 2.6. Isolation of the POCs
adjusted using NaOH solution (10 M) (from 3.2 – 3.5
to 7.0 – 7.2) after solubilization. Then, 2 ml tri-distilled According to Hassanpour et al.’s protocol , POCs were
[11]
water was added into 15% (w/v) gelatin and 3% (w/v) prepared from 4-week-old female Kunming mice and
sodium alginate (Sigma-Aldrich; Merck) and dissolved then encapsulated in the bioink. Briefly, each mouse
at 55°C for 30 min. Finally, dECM-based bioink working was intraperitoneally injected with 10 IU Pregnant Mare
solution was produced by mixing 3 ml dECM solution Serum Gonadotropin (PMSG, Solarbio) followed by
(pH 7.0 – 7.2) with the above 2 ml solution. 16 U Chorionic Gonadotrophin for Injection (Harbin
Sanma Animal Pharmaceutical Co. LTD) after 48 h, and
2.5. The biochemical characterization and then, the ovaries were isolated after 6 h. The ovaries were
biocompatibility of the dECM-based bioink incubated in α-MEM medium (Gibco) with 1% penicillin-
(1) Microarchitecture of the dECM-based bioink by streptomycin (PS, Millipore) at 4°C. Afterward, the
SEM ovaries were, manually, dissected with fine needles into
smaller fragments and incubated in a digestion solution
SEM was performed to observe the microarchitecture of consisting of Dispase II (Cat. No. D4693-1G, Sigma)
the bioink. First, the bioink was freeze-dried and then and Collagenase I (Cat. No. 1904MG100, BioFROXX)
fixed in 2.5% glutaraldehyde at 4°C for 12 h. Second, the at 37°C for 30 min. Then, the enzymatic process was
above samples were sequentially immersed in 70%, 80%, terminated by equal volumes of α-MEM medium
90%, and 100% ethanol for 15 min. The samples were containing 10% FBS (BI). The suspension was filtered
then coated with gold sputter and viewed under SEM. through a 100 µm cell strainer (Life Sciences, USA) and
washed twice with the following culture medium: α-MEM
(2) Circular dichroism (CD) spectra properties medium with 10% FBS, 3 ng/ml Recombinant Murine

To evaluate the protein structures and thermal stability Epidermal Growth Factor (Cat. no. 315-09, Pepro Tech),
of the bioink, we tested the bioink with CD, including 100 mIU/ml Follicle Stimulating Hormone for Injection

room temperature CD and the variable temperature CD (Harbin Sanma Animal Pharmaceutical Co. LTD), and
analysis. First, the baseline was measured, followed 1.5 U/ml chorionic gonadotrophin for injection and 1%

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