Page 280 - IJB-8-3

3D-bioprinted Ovary Initiated Puberty in the Model Mice
PS. Cells were resuspended in 200 µl culture medium 2.9. Functional evaluation
after centrifugation and then mixed well with the bioink (1) Neoangiogenesis and cell proliferation
for printing.
Three weeks after the surgery, vaginal smear was carried
2.7. Printing of POCs-laden structure and out with normal saline that was used to observe the change
culturing in vitro of each stage of the estrous cycle of the experimental
A porous circular grid 3D scaffold (nozzle diameter was mice. After 4 weeks of treating, the transplants were
340 µm, nozzle temperature was 20°C, and platform removed and routinely embedded them in paraffin and
temperature was 4°C) was printed using a bioprinting sectioned continuously. To evaluate the vascularization
system (Bio-Architect®-WS; Hangzhou Regenovo and cell proliferation of grafts in the above groups,
Biotechnology, Ltd.). During printing, the pneumatic IHC staining of a rabbit polyclonal antibody against the
pressure (0.18 – 0.32 kPa) was adjusted to match the best neovascular-specific cell surface markers CD31 (1:200;
nozzle scanning speed (6 mm). The bioink mixed with the Servicebio Technology Co., Ltd., Cat. No. GB11063-2)
POCs (1 × 10 cells/ml) was used to print. After printing, and a rabbit polyclonal antibody Ki67 (1:100; Wanleibio
6
the POCs-laden 3D scaffolds (2 mm in height and 5 mm Co.,Ltd., Cat. No. WL0280a) was performed. In addition,
in diameter) were soaked in 5% calcium chloride solution representative sections were stained with TUNEL
(3 min) for cross-linking. Then, the POCs-laden 3D following the manufacturer protocol (Servicebio™
scaffolds were cultured in an incubator (37°C, 5% CO ), Fluorescein Tunel Cell Apoptosis Detection Kit) to assess
2
and the POCs viability in the 3D scaffolds was tested the survival of POCs in vivo.
with a live/dead assay kit (Molecular Probes, Inc., Cat. (2) Sex hormones secretion
No. L3224) on the 1 , 7 , and 14 day. Then, the cells
st
th
th
were reviewed under a laser confocal microscope (Zeiss, Blood samples were collected by eyeball on day 28 after
Germany). transplantation. Then, the serum was separated overnight
at 4°C. All blood samples were, then, immediately stored
2.8. In vivo study: Female castrated mice model at −80°C until further analysis. The serum levels of
and grouping estradiol (E ), follicle stimulating hormone (FSH), and
2
progesterone (P) were measured using a competitive
The castrated mice model was performed as previously enzyme-linked immunosorbent assay kit (CUSABIO).
described . Briefly, the mice with a regular oestrum The sensitivity for E , FSH, and P kits was 40 pg/ml,
[9]
cycle phase were operated on both sides of the back 2.5 mIU/ml, or 2.5 mIU/ml, respectively.
2
1.0 cm incisions after being anesthetized successfully.
The incisions were made over the ovaries location (3) Expression of POCs markers
through the subcutaneous layers. Then, ovaries were
removed from the top of the uterine horns and sutured To assess the specific markers of the POCs on the
the skin at the incision site. Two weeks after the implanted grafts, immunofluorescence staining was done.
operation, the vaginal orifice of ovariectomized mice The sections obtained from the above were dewaxed to
was examined to determine whether the operation was water and rinsed in PBS (5 min). To reduce the non-
successful or not. The incisions on both sides of the specific background, the sections were soaked in 0.3%
back of the mice were reopened and bluntly stripped bovine serum albumin solution for 30 min. Then, the
to form a tunnel under the skin. The prepared grafts sections were stained using estrogen receptor alpha
were implanted, and the incisions were sutured with (ER-α, 1:200; Biosynthesis Biotechnology Co., Ltd.,
absorbable sutures (Shanghai Pudong Jinhuan Medical Cat. No. bs-0725R), progesterone receptor (PR, 1:200;
Products Co., LTD). Biosynthesis Biotechnology Co., Ltd., Cat. No. bs-
Twenty mice were randomly divided into five groups 23376R), inhibin alpha (Inhibin-α, 1:200; Biosynthesis
(four mice in each group): (i) Sham-operated mice as the Biotechnology Co., Ltd., Cat. No. bs-1032R), and anti-
non-ovariectomized (OVX) group; (ii) ovariectomized FSH-R pAb (FSHR, 1:200; Servicebio Technology Co.,
mice without further treatment as the OVX-C group; Ltd., Cat. No. GB11275-1). The secondary antibody used
(iii) ovariectomized mice receiving 3D scaffolds (8 × was Alexa Fluor® 488 Goat Anti-Rabbit antibody (1:200;
8 × 3 mm ) without cells as the 3D scaffold group; (iv) Invitrogen, CA), and counterstained with DAPI.
3
ovariectomized mice receiving 3D scaffolds with POCs 2.10. Statistical analysis
(1 × 10 cells/ml) as the 3D scaffold encapsulating POCs
7
group; and (v) ovariectomized mice receiving the bioink SPSS version 21.0 was used. Continuous variables are
with POCs without printing as the hydrogel encapsulating represented by mean ± standard deviation. The DNA
POCs group. content and IOD value were analyzed by a Student’s

272 International Journal of Bioprinting (2022)–Volume 8, Issue 3