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3D-bioprinted Ovary Initiated Puberty in the Model Mice
bioengineered ovaries with an appropriate cell- and consists of individual living cells or living cells with a
tissue-specific bioinks. supporting hydrogel component . At present, there is no
[15]
Bioengineered ovaries must mimic natural organs. published work on 3D bioprinting of ovaries using bioink
Besides isolated follicles, it also requires autologous composed of dECMs. However, 3D bioprinting should
ovarian cells, which are required for follicle survival . not be confused with 3D printing of biomaterials. The
[3]
The follicles are separated from the stromal elements by former refers to the printing process of live cells, while
encapsulating themselves in a basement membrane, and the latter refers to the printing of biomaterials, which
autologous ovarian cells are derived from cured ovarian does not require live cell printing (the printed scaffolds
samples . Thus, it can be considered a safer means of can be seeded with live cells) . The 3D-bioprinted
[4]
[16]
restoring fertility in women with cancer. cell-loaded scaffolds possess ideal spatial distribution.
Today, the materials used to construct bioengineered Thus, we hypothesize that 3D bioengineering ovarian
ovaries include both natural and synthetic polymers [5,6] . constructs using ovarian dECM-based bioink for tissue
However, studies reported that the matrix of choice for infiltration and target tissue remodeling will facilitate cell
artificial ovaries is the decellularized extracellular matrix distribution and survival.
(dECM) , because synthetic polymeric materials do In this study, we focused on 3D bioprinting porous
[7]
not possess every property of dECM . Ovary dECM cylindrical-shaped ovarian constructs employing swine
[8]
materials for bioengineering ovaries have produced ovarian dECM-based bioink encapsulating POCs to
some promising results. Laronda et al. first successfully evaluate the efficacy of ovarian failure correction.
[9]
constructed scaffolds from acellular bovine and human
ovarian tissues to support follicle growth and restoration 2. Materials and methods
of ovarian function in ovariectomized mice. Another
study produced a decellularized porcine ovarian matrix 2.1. Animals
that supported the survival of rat granulosa cells in One hundred and twenty slaughterhouse-raised female
vitro and improved estradiol hormone secretion . swines (95 – 100 kg, aged 6 months) were used to
[10]
Hassanpour et al. seeded rat primary ovarian cells harvest the fresh ovary tissues. Twelve female Kunming
[11]
(POCs) on the decellularized human ovarian matrix and mice (16 – 20 g, aged 8 weeks) were used to determine
found follicle-like structures within the matrix 4 weeks the biocompatibility of the dECM-based bioink. One
after transplantation. Pors et al. investigated human hundred and ninety female Kunming mice (13 – 15 g,
[12]
preantral follicles seeded on the human ovary dECM. aged 4 weeks) were used to prepare the POCs and animal
In vivo assessment showed that the survival of follicle model. All procedures involving animals were conducted
was higher in the decellularized human ovarian scaffolds in compliance with the guidelines of the local animal
after 3 weeks of xenografting in mice. Despite these ethics committee on animal care (No. 2019-P060).
promising results with ovarian dECMs, it is challenging
to find a precise fit with follicles of different sizes in the 2.2. Decellularization of ovarian tissues
pores of dECMs. An alternative approach is to convert
ovarian dECMs into a temperature-sensitive hydrogel. Fresh swine ovarian tissues decellularization was
[17]
This method can perfectly encapsulate isolated follicles conducted based on previous work . First, the ovaries
or other ovarian cells while maintaining acellular ovarian were cut into pieces (3 mm thick), cleaned with normal
tissue components. Chiti et al. converted bovine saline, and treated with phenylmethylsulfonyl fluoride
[13]
ovarian dECMs into hydrogel and demonstrated that (PMSF) solution (0.1 mM/L) in a shaker (130 rpm)
mouse preantral follicles were able to survive in the for 48 h at 4°C, which could inhibit protease activity.
hydrogel. Second, the tissues were placed into a hypotonic Tris
The traditional tissue engineering techniques buffer (pH 8.0) containing 0.1% sodium dodecyl sulfate
described above, such as seeding cells on dECMs or (SDS) and 0.1 mM/L PMSF for 12 h at 4°C. Third, the
encapsulating cells in hydrogels, can mimic physiological tissues were submerged in Tris-buffered saline containing
ovarian tissue and improve ovarian function to some 0.1 mM/L PMSF and 1% Triton X-100 solution by
extent. However, precise control of the spatial distribution, continuous shaking (130 rpm) at 4°C for 7 days. Fourth,
oxygen diffusion, or cell structure between cells and matrix the tissues were soaked in nuclease solution (pH 7.5)
remains a challenge for conventional tissue engineering. containing 50 U/ml deoxyribonuclease I (Sigma, Poole,
A solution to these problems are three-dimensional (3D) UK) and 1 U/ml ribonuclease A (Sigma, Poole, UK) by
bioprinting technology . 3D bioprinting technology has shaking (80 rpm) at 37°C for 12.5 h. Then, the tissues were
[14]
the potential to achieve ovarian morphological repair and immersed in 0.1% peroxyacetic acid and 20% ethanol at
reproductive endocrine function rebuild. The material 4°C for 2 h, and finally freeze-dried. The ovarian dECM
used for 3D bioprinting is called “bioink.” Bioink was then formed.

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