Introduction

The function of the female reproductive system can be permanently lost due to various congenital defects or acquired factors, resulting in infertility. These permanent dysfunctions are caused by various factors. Taking reproductive organ tumors as an example, of the existing treatment methods, such as chemotherapy, radiotherapy, and surgery,10 complete or partial resection has a greater risk, and radiotherapy and chemotherapy will damage the quality of the oocyte to a certain extent, resulting in oocyte being unable to mature normally or even dying, leading to premature ovarian failure and infertility.117,121,122,137 Postoperative tissue adhesion or fibrosis is also the most important factor affecting ovarian, uterine, or vaginal function, so one of the most important goals in treating cancer patients is to preserve fertility. Current methods of saving and preserving women's fertility include hormone therapy to restore ovarian function,39 freezing ovarian tissue,1 autogenous/allogeneic organ transplantation, in vitro maturation and fertilization of oocytes,1 and artificial ovarian transplantation.36 According to Chiti et al., more than 100 babies were born in vitro by cryopreservation and ovarian transplantation.22 However, frozen and transplanted ovaries are inapplicable to some patients, including patients with cancer metastasis and children. For example, adult ovarian tissue cryopreservation and transplantation are common in hematologic malignancies (Hodgkin lymphoma and leukemia) and breast cancer. Leukemia and myeloproliferative disorders are most common among children. However, transplantation means the risk of malignant cancer cells being implanted with transplanted tissue.8 For these reasons, researchers have been working on alternative methods to transplant isolated follicles and ovarian epithelial cells into artificial ovaries, allowing them to function as an ECM in the early stages of transplantation, as well as to perform endocrine functions, helping to develop follicles and temporarily or permanently replacing natural ovaries.3 The main purposes of using artificial ovaries are as follows: to safely transplant isolated primordial follicles and primary follicles, thereby preventing malignant tumor cell contamination in ovarian tissue and consequent disease recurrence; to support the survival and development of posttransplant follicles, and to ensure the secretion of sex hormones and the production of fertilized mature oocytes.

Ovary Structure and Follicle Development

The ovary is one of the most important organs in the female reproductive system, and it produces oocytes and performs endocrine functions. After birth and then puberty, the function of the ovary is to store oocytes in the follicles, providing suitable sites for follicle development and periodic oocyte release.6 After entering puberty, the endocrine function of the ovary begins to play a key role, and estrogen, testosterone, and progesterone begin to be secreted.109 A range of complex signals, including endocrine hormones, paracrine factors, ECM and mechanical signals, are exchanged between follicles and their environment.3 These highly dynamic interactions form the process of follicle growth, including recruitment, ovulation, and reproductive endocrine activity. The diameter of the primordial human follicles is 30–50 μm in the resting period, then approximately 100–200 μm when developing to the preantral follicles, and development toward antral follicles begins under the stimulation of FSH and LH.45,59,63 The diameter of the graff follicles and the preovulatory follicles can reach 18 mm, and the volume increases by approximately 600 times.5,47,48,49 To prepare artificial ovaries, an environment similar to that of natural ovaries must be engineered to promote follicle survival and growth. Ovarian follicles, which are the functional units of the ovary, comprise the ovarian parenchyma.85 Conceptualizing the stroma as the inverse of the parenchyma, the ovarian stroma thus refers to the components of the ovary that are not ovarian follicles. The ovarian stroma comprises general components such as immune cells,139 blood vessels,107 nerves,94 and lymphatic vessels,13 as well as ovary-specific components. These ovary-specific components (Fig. 1) include ovarian surface epithelium,7 tunica albuginea, intraovarian rete ovarii,135 hilar cells, ovarian stem cells,58 a majority of incompletely characterized stromal cells, and extracellular matrix (ECM).12 The ECM consists of fibril- and network-forming proteins, proteoglycans, and glycosaminoglycans. These diverse biochemical substances create a special ovarian structure and structural support for dynamic mechanics, and hydrogel provides exactly the kind of dynamic mechanical support needed for dynamic follicle development.8

Figure 1
figure 1

Components of the ovarian stroma.

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The Biomechanical Properties of the Ovary

The concentration of collagen in the ovarian stroma gradually decreases from the outer cortex to the inner medulla. The collagen fibers in the cortex are radial, making it relatively hard in terms of mechanical properties.56 The medulla layer is a porous structure composed of anisotropic collagen fibers. According to its weaker mechanical properties, it is stratified into a harder cortical layer and a less dense medulla layer. Early follicles grow and develop in the hard cortex. During further development, the follicles move toward the softer medulla and expand.101

In each reproductive cycle, the follicles develop, migrate, rupture and ovulate, and the ovaries undergo structural and functional tissue remodeling.92 Biochemical and mechanical signals regulate this process. Among them, matrix metalloproteinases (MMPs), MMPs inhibitors and plasminogen activators secreted by follicles and other ovarian stromal cells are important signal proteins that regulate the strength of the matrix,68 softening the ECM around the follicle to allow the follicle to expand.71 The ovary is a tissue with dynamic mechanical response, and the biomechanics of the ovarian matrix are essential for the development of follicles.114

Design and Main Preparation Routes of Artificial Ovaries

Design Element

The artificial ovary consists mainly of three parts: intact follicles, ovarian stromal cells, and biomaterials for the growth of supporting cells and tissue development. In terms of materials, the following three sets of requirements need to be met: ① The material must be nontoxic, with the ability to maintain cell vitality for a long time, allow enough gas exchange, nutrition diffusion, and intracellular water exchange, ensure normal communication between follicles and peripheral ovarian cells, and ensure follicle survival and development3,34,88; ②The material must have appropriate elasticity, which is directly related to follicle diameter, formation of follicular membrane, the formation of follicular cavity, secretion of estradiol, and the rate of meiosis recovery. The material needs a certain elasticity to allow the volume index expansion of the follicle during development, and must therefore have the appropriate mechanical properties and degradation rate to adapt to the increase of follicle volume, and to provide sufficient support for the morphological maintenance of its spherical shape to prevent the oocyte from peeling off; ③ the material needs to have the corresponding degradation rate to release the appropriate concentrations of growth factor, hormone, fetal globulin, transferrin, insulin, selenium and so on at the appropriate times.33,115 The performance requirements for artificial ovarian materials are shown in Table 1.128

Table 1 Ideal properties of biomaterials for artificial ovary.128
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Hydrogel Encapsulation to Prepare Artificial Follicles

The structure of the ovary consists of the ECM, blood vessels, follicles, and cells. The follicle is the main functional unit of the ovary and consists of follicular stromal cells, granulosa cells, and oocytes. Granulosa cells are mainly responsible for the production and metabolism of gonadotropins, estradiol and luteinizing hormone.9,125 The development of the oocyte is accompanied by the maturation of the follicle under the action of various hormones. Therefore, bionic artificial follicles that promote oocyte development and release from the follicle and restore reproductive hormone secretion and release have become an important breakthrough in preparing artificial ovaries. Conventional two-dimensional (2D) culture disrupts the close interaction between oocytes and surrounding stromal cells, thus preventing follicles from developing into antral follicles and reducing the follicle survival rate. Currently, the conventional approach to creating artificial ovaries is to encapsulate follicles in plasma clots,38 synthetic hydrogels,72 or natural polymeric hydrogels,138 such as collagen,21 fibrin,89,120 or alginate.61 Hydrogels with a three-dimensional(3D) structure that can mimic the ECM structure in vivo, provide mechanical support to the encapsulated cells and allow the exchange of oxygen, nutrients and metabolites by passive diffusion, maintain the formation of communication signals between oocytes and stromal cells, and facilitate the development of primordial follicles to sinus follicles, oocyte maturation, and ovulation, may become the main way to prepare artificial ovaries.

3D Printing to Prepare Artificial Ovaries

3D printing technology can be used to create biocompatible, biodegradable, and functional prosthetic organs or tissues, and has been used in regenerative medicine to prepare heart, dilated airway, and central nervous system scaffolds. With advances in 3D printing technology and cell biology, more functional organs can be created using new tissue bioinks. However, their use in female reproductive therapy is currently limited. Research into 3D printing systems for bioengineering reproductive tissues is critical for in vitro follicle culture, ovarian tissue transplantation, and menopausal hormone therapy. In 2017, Laronda et al. attempted to 3D print ovaries to partially restore ovarian function (i.e., hormone secretion and egg production). A 15 mm × 15 mm ovary scaffold was 3D printed using gelatin as the main material, and then added 40–50 follicles and hormone-producing cells to support the follicles in the 3D ovary. This research successfully produced young mice.81 Additionally, work by Raffel et al. found that interconnected pore networks fabricated by electrostatic spinning of polycaprolactone (PCL) were also able to maintain the 3D structure of porcine follicles.105 Wu et al. used gelatin-methacrylamide (GelMA), a bioink, to 3D print biocompatible and mechanically suitable artificial ovaries. Exogenous follicles were also deposited in the scaffold and over time resulted in mature oocytes.138

3D printed ovaries are a great advancement at the intersection of three disciplines (manufacturing technology, materials science and reproductive medicine), and are one of the exploratory cases of using regenerative medicine to address human diseases. However, the clinical application of 3D printed ovaries is still difficult due to the large differences between mouse and human species. First, human ovaries are far larger and more structurally complex than those of mice,66 which places a higher demand on the variability of ovarian traits. The materials currently used are not strong enough to support the human ovary. Second, in infertility patients encountered in the clinic, the ovaries are often not fully functional.15 The use of 3D printed ovaries does not achieve a real technological breakthrough on the bottlenecks and difficulties of regenerative medicine. The real difficulty in regenerative medicine is now the regulation of the microenvironment and how to follow a set program to grow into the expected tissue or organ in vivo without interference. This problem is currently unresolved worldwide. Although 3D printed ovaries are still a long way from clinical application, the development of 3D printing technology has given regenerative medicine researchers a new idea for in vitro organ construction.

Natural Polymeric Materials Commonly Used in Artificial Ovaries

Most of the repair and regeneration materials used in reproductive regenerative medicine are natural polymer materials derived from animals or human tissues, including alginate, gelatin, collagen, fibrin and hyaluronic acid.128 There are also synthetic polymer materials such as polyethylene glycol,29 dimethione77 and silicon dioxide. Natural polymeric materials are similar to the human ECM and its bioactive factors. It plays an important role in cell adhesion, migration, proliferation and differentiation with good biocompatibility.11 Synthetic polymers have excellent mechanical properties, including good plasticity, and are suitable for specific application scenarios. For example, hydrophilic polymers can be used to make hydrogel scaffolds.96 However, compared to natural polymers, some synthetic polymers can suffer from poor cytocompatibility,52,78,116,132 limited biocompatibility87 and lack of hemocompatibility,50,134 limiting their use in specific areas.112 The applications of natural polymer materials in artificial ovaries are described below.

Alginate

Alginate is a natural polysaccharide carbohydrate extracted from kelp and consists of (1 → 4)-β-cross-linked d-mannituronic acid and (1 → 4)-α-cross-linked guolinic acid. Alginate solution forms a gel in the presence of Ca2+, and its hydrogel has good biocompatibility, biodegradability, nontoxicity and nonimmunity, and becomes an ideal material for the preparation of tissue engineering scaffolds.4 It can provide a better growth environment for the development of human primordial follicles when it is used as an ovarian scaffold, and can complete allografts. The hardness of alginate can be regulated by its composition or concentration to enable the survival and development of follicles and the maturation of oocytes.17 The production of secreted hormones has a certain promoting effect.133 Jiao studies have reported that the hardness of the alginate matrix can affect the expression of oocyte genes, and appropriate hardness is beneficial to the expansion and growth of oocytes, the proliferation of granulosa cells and the formation of the follicular cavity.64 The secondary follicles of nonhuman primates secrete VEGF-A, ANGPT-1 and ANGPT-2 in 0.25% alginate.41 Rodrigues found that they also secrete steroids and anti-Mullerian hormone. Concentrations of 0.125%–3% alginate were studied. A high concentration of alginate (0.5%–1%) can improve follicle morphology. A low concentration of alginate (0.25%) is beneficial to the production of estradiol and progesterone.103 For human small antral follicles, a concentration of 1% or 1.5% alginate is most appropriate and this concentration range supports follicle survival and growth. Different alginate concentrations lead to different follicular development results because the concentration of alginate affects its biomechanical properties. Primitive follicles need a hard environment similar to the ovarian cortex, equivalent to 2% alginate. Late developing follicles require a less rigid environment, equivalent to 0.25% alginate, making it easier to squeeze out the first polar body.98

In recent studies, scientists no longer satisfied with in vitro culture and the development of follicles in alginate artificial ovaries have instead studied the physiological effects of alginate-based artificial ovaries in vivo, such as the secretion of cytokines and hormones. Kreeger et al. prepared culture systems by combining calcium alginate with a 3D acellular ovarian matrix. Arginine-glycine-aspartate peptides was grafted on the acellular ovarian matrix.76 Sittadjody et al. used 3D bioengineering methods, in which granulosa cells were encapsulated in 1.5% ultrahigh purity and low viscosity mannitoluric acid microcapsules, and coated with 0.1% polyornithine (PLO). PLO coated microcapsules were then mixed with stromal cells suspended in 1.5% ultra-pure low-viscosity manulonic acid, and then sealed with a microfluidic device. The double-layer alginate micro capsules were prepared using Ca2+ and Sr2+ to cross-link alginate. A layer of 0.5% ultra-pure and low-viscosity guroglyoxalic acid was sprayed on its periphery. The outermost PLO and alginate layers held the stromal cell layers used to prevent an immune response. A model of cell-based clinical hormone replacement therapy (cHRT), structure is shown in Figure 2. Animal experiments have shown that this artificial ovarian structure can effectively alleviate the hormone loss caused by ovariectomy. Plasma osteocalcin and CTx (C-terminal peptide) are continuously expressed, and this artificial ovary can relieve ovarian failure.119

Figure 2
figure 2

(a) Schematic diagram of artificial ovary constructed by Sittadjody; b. confocal microscope PLO (poly (ornithine), TC (stromal cells), GC (granulosa cells) green: granulosa cells; red: ovarian stromal cells. Copyright (2017) Nature publication.119

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Although alginate is widely used in tissue engineering scaffold materials, because of its strong hydrophilicity, a gel made of alginate alone is not easy to degrade. With the growth and diameter expansion of follicles, the compression force acting on follicles increases, which is not conducive to follicle expansion. Moreover, spindle assembly during the meiosis of oocytes may be disturbed by alginate gel,90 which restricts its application as a single material in artificial ovaries.

Fibrin Protein

To mimic the dynamic biomechanics of the ovaries, Ariella Shikanov combined degradable fibrin and nondegradable alginate to prepare a fibrin-alginate interaction network (FA-IPN) hydrogel (Fig. 3). Interaction networks (IPNs) are network structures in which polymers with independent molecular chains are combined with chemical or nonchemical bonds. In FA-IPN hydrogels, fibrin is biologically active and can be gradually degraded by proteases secreted by follicles to free up enough space for follicle expansion, while alginate continues to support the integrity of follicular structure. FA-IPN materials possess dynamic mechanical properties consistent with the follicle requirements for growth and development. With the development of follicles, fibrin gradually degrades, the elastic modulus gradually decreases, and the resistance to follicle expansion becomes smaller. Fibrin and alginate can gradually form gels under neutral conditions, which is beneficial to cell encapsulation, and their mechanical properties can also be regulated by the content of each component. FA-IPN promotes follicle development and increases the number of meiotic oocytes.126 Therefore, fibrin-alginate hydrogel and fibrin-alginate-matrix glue can be used in the in vitro culture of ovarian cells and follicles.

Figure 3
figure 3

FA-IPN electron microscopy. A. Fibrin gel containing 5 U/mL thrombin; B.FA-IPN containing 5 U/mL thrombin; C.FA-IPN containing 50 U/mL thrombin; D.FA-IPN containing 500 U/mL thrombin. Bar = 3 μm, Copyright (2014) Wiley Online Library.126

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Fibrin scaffolds are the best choice in some studies of in vivo artificial ovarian models. The basic structure of fibrin consists of fibrinogen and thrombin. Fibrin not only plays an important role in the coagulation process, but also has other functions, such as involvement in intracellular and matrix interactions, inflammatory responses, wound healing, and fibrinolysis.95 Therefore, it can be used as a tissue engineering scaffold in the clinic, such as for making artificial ovaries. Fibrin clots can be used to recruit ovarian membrane cells, but their disadvantages are also obvious. The degradation rate is too fast, and because of the relatively low elastic modulus, there is a lack of sufficient physical support before cell infiltration and proliferation. To better control its degradation performance, the concentration of fibrinogen and thrombin can be increased, or other natural or synthetic polymers can be added. Its hardness can also be adjusted by changing the fibrinogen/thrombin (F/T) concentration or by mixing with other materials. Therefore, it is crucial to find the optimal ratio of fibrinogen and thrombin, and to regulate scaffold porosity and hardness (Fig. 4). A fibrinogen and thrombin matrix at a low concentration (F12.5/T1) was suitable for secondary follicle encapsulation and transplantation in mice,24,26 but lacks sufficient hardness to support the development of primordial and primary follicles. When the concentration ratio was increased to F50/T50 and F75/T75, the resulting constructed ovaries were almost as thick as human ovarian cortical fibers. However, increasing fibrinogen and thrombin not only increases the follicle storage, but increases the hardness as well. By comparison, the F50/T50 ratio presents the best microstructure and hardness, to reconstruct the human ovarian cortex most closely.25

Figure 4
figure 4

Electron microscopy image of artificial ovaries prepared with different fibrin and thrombin concentrations and ratios (×2000, ×12,000, ×90,000), Copyright (2017) Springer.25

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Songsasen et al. found that a fibrin-alginate matrix is more beneficial to the development of canine secondary follicles than pure alginate, probably because the combined use of fibrin and alginate modulates the degradability and mechanical properties. The degradation properties of the matrix play an important role in enabling the successful cultivation of preantral follicles of goats and dogs in vitro. Artificial ovarian models made from fibrin-alginate and matrix glue have also been used in nonhuman primates to develop preantral follicles in vitro into antral follicles. Although the fertilization rate of oocytes was relatively low, this study demonstrated for the first time that a fibrin matrix with its dynamic mechanical properties is suitable for the in vitro culture of nonhuman primate preantral follicles.77 Xu et al. compared the effect of a fibrin-alginate matrix and a pure alginate matrix on the development of primordial follicles, primary follicles, and secondary follicles in macaques, and found that the development rate of primordial follicles in a fibrin-alginate matrix was higher than that of pure alginate, but secondary follicles developed better in alginate.141

A team of researchers at the University of Leuven in 2017 conducted research on the 3D structure of degradable fibrin artificial ovaries. Studies have shown that fibrin with low fibrinogen and thrombin concentrations can be better used in the construction of biodegradable and biocompatible 3D matrixes to enable the survival and proliferation of ovarian stromal cells. In all the different fibrin matrix concentration tests, the F50/T50 concentration of fibrin in the ultrastructure and stiffness was the best combination,67 closest to the human ovarian cortex.22,25 Additionally, fibrin can cross-link through different growth factors and angiogenic factors, stimulate neovascularization through a controlled release mechanism during fibrin degradation, and reduce hypoxic damage without vascular transplantation. Despite its low elastic modulus, its support hardness can be easily changed by changing the F/T concentration or mixing it with other materials.25 Although fibrin has enabled significant progress in the regeneration of the female reproductive environment, other requirements, such as matrix hardness and hardness regulation, and the rapid infiltration of vascular endothelial cells, must be met to reconstruct the optimal in vitro and/or in vivo environment during follicle survival and development.

Gelatin

Gelatin is a collagen hydrolysate extracted from the bone, skin and connective tissue of horse, pig, chicken, and fish. Gelatin lacks antigenicity due to thermal denaturation, so it does not easily to cause an immune response and has good physical and chemical properties, biocompatibility, and biodegradability. Moreover, gelatin can form a porous scaffold material after processing, and the pore size can be artificially adjusted to make it an ideal tissue engineering scaffold material. Its application scope includes tissue engineering, such as cartilage, blood, vessels, tendon, and mucous membranes, and it is also used to prepare drug carriers. In the field of assisted reproduction, gelatin can be used in the in vitro culture of human follicles. Laronda et al. invented 3D-printed gelatin scaffolds based on the porous nature of the ECM. Follicles were implanted in the gelatin scaffold, and somatic cells followed the follicle edge and adhered to the scaffold. The survival rate of oocytes increased as the number of follicles adhered to the stent increased.128 Most 3D culture systems use serum-free medium, alginate gel, or collagen to encapsulate in vitro cultured oocytes.128 These methods provide adequate physical support for follicular development, but granulosa cells and stromal cells are unable to perform endocrine and paracrine functions, causing follicular development to fail, so blood circulation and hormone secretion within the graft must be considered. Moreover, the brittleness of a gel made of gelatin alone causes frequent ruptures. Researchers have made many attempts to solve these problems.

Woodruff et al. invented a gelatin scaffold (Fig. 5a) that allows gelatin to self-reinforce without rupture at approximately 30 °C, resulting in a multilayer structure. This geometry is directly associated with whether the follicle (a structure formed by immature oocyte cells surrounded by hormone-producing granulosa cells) can survive in the ovary. Woodruff et al. removed mouse ovaries and implanted 3D-printed gelatin scaffold ovaries, which contained immature oocytes. The porous structure of the scaffold provided space for oocyte cell maturation, ovulation, and the formation of blood vessels within the scaffold, promoting the survival of immature oocyte cells and hormone-producing granulosa cells in mice (Fig. 5b), not only enabling these gelatin scaffold ovarian transplanted mic, but also successfully promoting hormone production in mice, producing hormones that circulate in the blood with many new blood vessels and promoted milk production in healthy young mice after delivery.81 In future, 3D printing is expected to be used to construct human ovarian scaffolds for further progress.

Figure 5
figure 5

(a) Schematic of thermally reversible gelatin cross-linking; (b) light microscopy image of oocytes that survived and developed in a gelatin scaffold for 8 days, Copyright (2017) Nature Publishing Group.81

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Agarose

Agarose, the main component of the cell wall of red algae, exhibits biological safety and degradability. Using its gelation phenomenon, agarose can be made into a 3D network structure and has been widely used in biomedical research fields, such as nerve regeneration, and bone regeneration scaffold. Krotz et al. announced in 2010 that his group had successfully grown the world's first artificial ovary in the laboratory (Fig. 6) that allowed human oocytes to mature in vitro.77 The team poured 3 mL of 2% sterilized ultra-pure electrophoresis grade agarose solution into a PDMS mold, and after 15 min, the cured agarose gel was removed from the PDMS mold. Three types of ovarian cells (follicular membrane cells, granulosa cells, oocytes) were placed in the molded structure composed of plastic agarose gel designed to mimic ovaries in vivo for 3D culture. The follicular membrane cells were cultured in a honeycomb-like structure, and then the cumulus complex was implanted into the structure. The granulosa cells aggregated and formed dense cumulus complexes within 48 h. After 72 h, one of the oocytes emerged from the cumulus cells and excreted the first polar body. The artificial ovary will allow mechanically isolated primordial oocytes or ovarian cortex to continue to develop and mature (without granulosa cell expansion or follicular membrane cell recruitment). Unlike alginate scaffolds, because follicular membrane cells and granulosa cells were involved in the construction of this artificial ovary, they can continue to produce hormones throughout the development of oocytes. This method is the first successful use of 3D tissue engineering to allow oocytes to mature in vitro. The breakthrough has had a significant impact on fertility research to help women facing chemotherapy or other treatments to retain fertility.

Figure 6
figure 6

A honeycomb artificial ovary prepared by agarose gel used for in vitro culture of the granulosa cells and oocytes; Copyright (2010) Springer.77 (a) Isolated cumulus oocyte complex; (b) Cumulus oocyte complex transplanted into artificial ovary model and cultured 22 h; (c) cumulus oocyte complex transplanted into artificial ovary model and cultured 46 h.

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Acellular Ovarian Matrix

The ECM of ovarian tissue consists of a variety of insoluble proteins and glycoproteins, including collagen, laminin, fibronectin, and glycosaminoglycan, and endogenous active factors, including TGF, FGF, and VEGF.12 The acellular ovarian matrix plays an important role in ovarian function by participating in follicle development, granulosa cell proliferation, and steroid production. Hassanpour et al. described in detail the process of ovarian decellularization: dividing the ovary into two, and cutting it into strips. Then, approximately 2.0 mm thick cortical tissue sections were isolated from the cortex. The remaining fragments were centrifuged for 48 h at speeds of 100 RPM and 500 RPM at 20 °C with 1% SLES (Kimia Sanaat Ataman Co., Tehran, Iran), then incubated with 500 U/mL DNase I (Sigma-Aldrich, Gillingham, UK) for 24 h in 36 °C PBS and rinsed several times in PBS to remove cell residues and chemical reagents.54

Laronda et al. was the first to successfully construct a bovine ovarian acellular scaffold and a human ovarian acellular scaffold in 2015, and to implant mouse primary ovarian cells followed by orthotopic transplantation to the ovarian location of ovariectomized mice.128 These recellularized scaffold materials can partially restore endocrine function in ovariectomized mice and induce puberty. However, this decellularization approach has some limitations. First, the long-term use of SDS has a serious effect on the ultrastructure of scaffold materials, so reducing the incubation time in SDS can improve the preservation of the ECM. Liu et al. replaced the SDS with Triton X-100 to reduce the chemical exposure time, added DNase I enzymes for treatment, increased the number of freeze–thaw times to induce cells to dissolve quickly, and promoted the penetration of compounds. Its acellular ovarian scaffold material can support cell adhesion and proliferation and the maintenance of E2 secretion. Liu transplanted rat ovarian tissue into a to the porcine acellular ovarian scaffold, and evaluated the graft tissue regeneration ability. The results showed that granulosa cells could enter the acellular ovarian scaffold for growth and improve their endocrine function.79

Jakus et al. reported the first organ-specific cell scaffold in 2017.42 The team prepared six different scaffolds for cell growth and wound repair, including a scaffold composed of 35% biocompatible polymer PLGA and 65% organ-specific ECM, and generated ovarian tissue flakes (Fig. 7). Ovarian tissue was decellularized in 0.5% SDS, after washing, freeze-drying, freezing and grinding to produce acellular ovarian powder, and then suspended in a three-solvent mixture of dichloromethane (DCM), 2-butoxyethanol and dibutyl phthalate (plasticizer), and dissolved in PLGA (82:18 lactic acid and glycolic acid; 35% solid) to produce acellular ovarian ink. The obtained acellular ovarian ink was immediately cast into a flat-bottomed container and dried at room temperature. After 15 min, the water had evaporated completely, and approximately about 40 cm2 of porous, highly microscopic and nanostructured ovarian flake tissue (OTP) was obtained for every 0.3 g of acellular ovary. Transplantation of isolated mouse secondary follicles onto the OTP and in vitro culture revealed that the OTP supported mouse ovarian follicle attachment and survival. The human and rhesus monkey follicles were then cultured in OTP with a 6 mm aperture. The histology and hormone secretion results after 8 weeks showed that the follicular survival rate exceeded 46.7%, and the secretion of estradiol was detected in the culture medium.42

Figure 7
figure 7

Preparation of bovine acellular ovarian tissue slice and electron microscopy, Copyright (2017) Wiley Online Library.42

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Although the use of acellular ovarian stroma is promising, it is difficult to find a suitable pore size matrix considering that the diameter of the preaural follicle is approximately 30–150 μm. The preparation of the matrix as gel as a feasible method enables encapsulation of isolated follicles and ovarian cells and retention of the components of the acellular ovarian matrix. Hydrogels based on ECM (ES hydrogels) can promote the reformation of functional tissues because their binding can maintain biological activity and promote tissue regeneration.74 The advantage of this type of hydrogel is that cytokines and related proteins retained in tissues after decellularization are preserved, and these cytokines can promote tissue growth and be released into regenerated tissues as the hydrogel degrades. Moreover, the stiffness of the ES hydrogel is suitable for the development of follicles. Follicles cultured in 2D in vitro destroys the communication between cells and directly affects the gap junctions between oocytes and granulosa cells. However, ES hydrogels have low stiffness and can promote the exchange of nutrients and hormones. Jungkin et al. used ES hydrogel to 3D culture follicles in vitro and compared it with alginate hydrogel. The mechanical properties of ES hydrogel and alginate hydrogel are different. The storage modulus of alginate is lower than 1000 Pa, while the storage modulus of the ES hydrogel is lower than 100 Pa, and the stiffness of ES hydrogel is lower than that of alginate. The results showed that the formation of ES hydrogel in the follicular cavity, the formation of COC, the maturation of oocytes, and the development of normal chromosomes and spindles were better than those obtained with alginate. The results show that compared with alginate, follicles can be better cultured in vitro in ES hydrogels.74

Hyaluronic Acid

Hyaluronic acid (HA) is an anionic nonsulfated mucopolysaccharide that is widely distributed in epithelial and connective tissue and promotes the regeneration of cells or tissues. Desai et al. encapsulated mouse follicles in a tyramine-based HA hydrogel for 3D culture.32 HA in the range of 2–5 mg/mL was prepared with acellular ovarian matrix at a ratio of 9:1 and inoculated with follicles for gel encapsulation. The results showed that the follicles with intact oocytes increased in diameter and remained spherical, which supported the recovery of meiosis and enabled estradiol secretion.

Parisa et al. prepared a hyaluronic acid-alginate (HAA) hydrogel to coculture mouse ovarian follicles, and compared the results with those obtained using alginate (ALG) and fibrin-alginate (FA) hydrogels. In the absence of OCs, higher numbers of ALG- and HAA-encapsulated follicles reached the antral stage than FA-encapsulated follicles. However, a higher percentage of HAA-developed oocytes resumed meiosis up to the germinal vesicle breakdown (GVBD)/metaphase II (MII) stages than ALG-developed oocytes. HAA-encapsulated follicles had significant overexpression of most of the growth and differentiation genes, and secreted higher levels of estradiol (E2) compared to ALG- and FA-encapsulated follicles. The study results showed that HAA hydrogels are a promising hydrogel for follicle culture due to their suitable stiffness.62

Chitosan

Chitosan (CS) is a promising material for artificial ovarian scaffolds due to its biocompatibility, biodegradability and inherent bioactivities, such as antibacterial, antioxidant and anti-inflammatory activities.65,30 Hassani et al. first reported the use of different concentrations of chitosan hydrogels for follicle culture. CS (0.5%) had a relatively high swelling rate and weak mechanical properties. The pores of 1.5% CS were not suitable for follicle development. However, 1% CS hydrogels with uniformly distributed pores and a higher degradation rate were the best concentration for follicle culture. The elastic modulus was 19.8 kPa and the shear modulus was less than 250 Pa and less than that of 0.7% alginate (203 Pa). Antral formation, survival and MII rates, follicle diameter, estradiol secretion, normal appearance of the meiotic spindle and chromosome arrangement were higher in the 1% CS group than in the 0.7% alginate group on days 6 and 13.53 However, both CS hydrogels and alginate hydrogels impaired oocyte complex detachment and oocyte maturation, which may be related to the mechanical properties of the culture medium and hydrogels. CS hydrogels have low viscoelastic properties and high stiffness, which hinder their further application in cell encapsulation. SF has higher flexibility. Jafari et al. synthesized a hydrogel with CS and SF using horseradish peroxidase (HRP) and hydrogen peroxidase-promoted crosslinking, and by varying the SF concentration, it was possible to modulate the viscoelasticity of the hydrogel to achieve similar viscoelasticity to the ECM and a similar shear modulus of normal ovaries to promote the proliferation of ovarian stromal cells.60

Others

Collagen I is a common substance in ovarian ECM, that is rich in glycine and proline and is present throughout the ovary. The expression of collagen I is the highest in the epithelial and follicular regions of the ovarian surface. 3D scaffolds constructed from collagen I can be used to culture granulosa cells and transplanted into the ovarian sacs of immunodeficient mice. Yang et al. dissolved collagen I in 1 mL of PBS to prepare a gel scaffold. UC-MSCs were inoculated into a gel scaffold to prepare an injectable gel scaffold, which was then injected into the ovarian medulla of POF mice. After injection, the mice exhibited an estrus cycle, increased estradiol and AMH secretion, promoted ovarian development, and granulosa cell proliferation, while ovarian function was preserved and POF symptoms were relieved.89

Table 2 summarizes the main findings obtained after grafting isolated follicles into the abovementioned matrices.

Table 2 Efficiency of different matrices used to graft isolated mouse or human follicles.35
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The Main Problems that Need to be Overcome and Countermeasures

How can Blood Recirculation be Reestablished?

Short-term posttransplant ischemia and hypoxia are among the reasons for ovarian transplantation failure. Neointima formation occurred within 48 h after transplantation in rats and 7 and 5 days after transplantation in sheep and humans, respectively. As with natural ovarian transplants, many follicular deaths after transplantation are directly related to ischemia during the initial postimplantation phase,16,31 limiting the longevity and success rate of artificial ovarian transplants. Many studies have attempted to shorten the duration of ischemia and promote neovascularization. Researchers have successfully delivered litters in mouse models with smaller ovarian sizes by encapsulating isolated follicles or ovarian tissue in hydrogel material containing growth factors or vascular endothelial growth factors for in vivo transplantation. However, in larger animals, such as pigs, monkeys, and even humans, the follicles are more susceptible to hypoxia and ischemia due to more fibrotic tissue and reduced follicular density.73

Blood vessels are the only way to obtain the nutrients and hormones needed for follicular development, and inadequate blood supply can limit follicular development leading to follicular atresia.14,43 Therefore, short-term vascular regeneration is crucial for the survival and regeneration of transplanted tissue.131,143 The design of materials such as microchannel network hydrogels and stem cell therapy is relatively new and effective method to rapidly establish blood recirculation in the material and medical fields.

Microchannel Network Hydrogel

To provide sufficient oxygen and nutrients to tissue engineering constructs, many research efforts have been made to mimic the luminal structure of blood vessels by creating microvascular-like channels in the constructs.86 Jung developed a microchannel network hydrogel composed of gelatin with perfusive properties to treat ischemic diseases by promoting angiogenesis, monocyte infiltration, and macrophage M2 polarization. To mimic the diameter range of capillaries (5–10 μm), the microchannel network in this hydrogel is 16.37 ± 7.76 μm in diameter and can connect to adjacent growing host vessels, allowing reperfusion of the entire vascular network of the hydrogel implant.82 When microchannel hydrogels were implanted in animal models of hindlimb ischemia in mice and pigs, the animals' blood vessels were connected to the hydrogel microchannels and perfused. Hindlimb ischemia in the experimental animals was alleviated. This suggests that endowing the material with porous microchannels can accelerate the recirculation of blood. Takei et al. used alginate hydrogel microfibers to assemble vascular-like microchannels.83 Their study found that cell culture fluid could flow rapidly (within 10 min) through the channels after immobilizing the cells in the assembled gel. Human hepatoblastoma cells proliferated in the gel portion of the microfibrils and maintained their specific functions during 7 days of perfusion culture.84

Vascularization is critical for the survival of implanted tissues in vivo, and the physical structure of channels provides an entry point for vascular growth. Mao et al. created three microchannels 1 mm in diameter in polyethylene glycol diacrylate (PEG) hydrogel cylinders. In vivo subcutaneous implantation experiments showed no significant host tissue infiltration in the PEG hydrogel without microchannels. The PEG hydrogel with three microchannels showed significant host tissue infiltration in the lumen. The infiltrated tissues included vascular structures with erythrocyte filling, and the arrangement of vascular endothelial cells was observed. This study directly demonstrates the importance of microchannels in the hydrogel construct for vascularization in vivo.124,127 Rapid vascularization is a crucial challenge for the survival of artificial ovaries and the flow of hormone secretion. Channels act as a biological pathway to facilitate nutrient transport and rapid waste removal, thereby accelerating cell growth and blood vessel formation.51 Many studies have focused on embedding microchannels into cross-linked hydrogels, which are biocompatible and malleable to encapsulate cells. Based on these advantages of hydrogels, the incorporation of channels further enhances the function of hydrogels in vascularization.93

Stem Cell Therapy

Stem cell therapy has shown great potential in regenerative medicine, and transplantation therapy based on mesenchymal stem cells (MSCs) has been shown to improve ischemic perfusion and circulation.57 In addition to differentiating into endothelial cells, MSCs can participate in angiogenesis by secreting angiogenic paracrine factors, including VEGF, bFGF, and PDGF,99,129 which have been studied extensively. Intramyocardial injection of MSCs for the treatment of myocardial infarction showed that patients had increased local blood volume and reduced ischemic area, which contributed to angiogenesis. In the field of reproductive health, stem cells have been used to treat ovarian dysfunction. Placental mesenchymal stem cells (PD-MSCs) have a role in promoting angiogenesis, inducing the upregulation of VEGF and VEGFR2 and activation of their signaling pathways in damaged ovarian tissue, promoting vascular remodeling in the ovary and ultimately follicle development. Thus, it is clear that stem cell therapy may be one of the solutions for blood recirculation after artificial ovarian implantation.27,97,113

How can Hormone Synthesis and Release Channels be Established?

The synthesis of ovarian hormones by the follicle, which play a crucial role in maintaining the ovarian cycle, determining secondary sexual characteristics, and preparing the endometrium for implantation, involves intercellular communication between the two main types of endocrine cells in the follicle40,118,136 (granulosa cells and luteinizing cells). The circulation of blood in the body is the primary way to provide hormones under normal conditions. Until the circulation of blood is established, the release of hormones through the material to mimic intercellular communication is the only way for the follicle to receive developmental signals, which are necessary for the further development of normal follicles. Currently, there are two methods to release hormones from materials. The first is to encapsulate cytokines and related hormones directly in materials and implant them in the body, releasing them through material slow release. The second is cell-based HRT (cHRT). Unlike the pharmacological approach, certain physiological outcomes can be achieved at lower circulating hormone levels due to the involvement of cells in the hypothalamic-pituitary-ovarian feedback control loop.

In Situ Implantable Controlled Release Drug Delivery

Biomaterials, such as nanospheres, and injectable hydrogels have been used in studies on the slow release of hormones, which are highly bioavailable after implantation for the constant release of hormones through the in situ controlled release of the material.70 The degradation of the materials can also improve the effectiveness of controlled release systems.69 Among synthetic polymers, PLA, PGA, and PLGA have good biodegradability, biocompatibility, and mechanical strength, and have been successfully used for nanoparticle-based drug delivery. Among natural polymers, cellulose, chitosan, silk, and HA are often used as hydrogels for drug delivery because of their excellent biocompatibility, biodegradability, and noncytotoxicity.100,123

Cell-Based Hormone Replacement Therapy

Current hormone replacement therapy (HRT), which is primarily based on estrogen and progesterone, has been prescribed for decades to treat postmenopausal symptoms. However, it has been questioned due to its side effects (venous thromboembolism, stroke, gynecological cancers),18,19,55,91,108,111 and is not suitable for premenopausal women.2 Along with releasing eggs, ovaries secrete several hormones, including oestrogen, progesterone, testosterone, activin, inhibin, anti-Müllerian hormone (AMH) and insulin-like growth factor 1 (IGF-1), which have an immense significance to a woman’s physiology.2

Cell-based hormone replacement therapy (cHRT) is a novel approach to enable the, physiological delivery of all ovarian hormones without causing any side effects.119 This concept of designing an artificial ovary involves dissecting all the cells in the ovary and subsequently amalgamating them. Paolo et al. encapsulated ovarian cells in a silica 3D matrix for in vitro culture, during which the luteinizing cells and granulosa cells communicate effectively, the nutrients and growth factors encapsulated in the silica matrix can diffuse into the follicles, and the hormones synthesized by the cells can be released normally.77 Sittadjody et al. devised an in vitro tissue-based hormone delivery technique using a multilayered functional construct fabricated using encapsulation technology. They integrated luteal and granulosa cells isolated from rat ovaries and used multilayer alginate microcapsules to mimic the natural follicular structure. The encapsulated cells secreted sufficient levels of estradiol, and progesterone in response to follicle-stimulating hormone (FSH) and luteinizing hormone (LH). It was shown that this approach leads to the secretion of sex steroids and peptide hormones, and responds to gonadotropins.119

Microfluidics

Microfluidic culture systems allow for the easy coculture of cells, and the addition or removal of substances needed for studies, such as pituitary or sex hormones, to mimic the dynamic hormone levels required for downstream tissue function, timely delivery of oxygen and nutrients, and timely removal of waste products.142 Microfluidic systems have also been applied in the field of reproductive organs. The multiorgan microfluidic system developed by Xiao et al. can support ovarian follicular tissue to generate menstrual cycle hormone profiles at day 28 of in vitro culture, and the system can provide a platform for endocrine cycle studies, such as the hormonal signaling involved in the menstrual cycle and pregnancy.119 Choi et al. used alginate and collagen to synthesize ovarian microtissues with ovarian cortex, and applied them to the miniaturized 3D culture of early secondary antral follicles.28 The development of bionic ovarian microtissue and microfluidics in this study is important for improving the quality of follicles cultured in vitro and understanding follicular development and ovulation mechanisms.44,140

Conclusion

The success rate and popularity of oocyte freezing and resuscitation technology are significantly higher than those of artificial ovaries, for recreating fertility, but artificial ovaries have three irreplaceable advantages for future development: (1) they can be used for ovarian tissue transplantation to maintain female fertility; (2) they can maintain low or, even normal functioning of the endocrine system through cell proliferation and tissue regeneration and present an alternative to hormone therapy for diseases associated with postmenopausal women; and (3) they can serve as a tool for testing follicle physiology and toxicology in preclinical drug development. To date, researchers have investigated several different treatments and biomaterials and their preparation methods to construct artificial ovaries and evaluate the recovery of ovarian function, such as ovulation and estradiol secretion. The design and development of future artificial ovarian biomaterials will require more consideration of ovarian function, such as oocyte maturation, ovulation, luteal formation, and progesterone secretion and release. After overcoming the recovery of artificial ovarian function, one of the greatest challenges in the future will be to construct a structurally firm, stable, and durable artificial ovary to conduct monitoring at multiple time points and more longitudinal studies in vivo for analysis of the reproductive and endocrine capabilities of artificial ovarian tissue and maintain it for 10 years or more. The second challenge is whether complete artificial ovarian scaffolds can be recellularized, such as by using ovarian cells or ovarian stem cells isolated from fresh or cryopreserved ovarian tissues to induce multifunctional stem cells or minimal embryo-like stem cells to refill artificial ovarian scaffolds.67 The third challenge is how to maintain the gap junction between oocytes and granulosa cells of large animal species in the artificial ovary and promote follicle expansion and oocyte maturation to create desired effects in vitro culture systems.

The author has no conflict of interest.